HLA-DR-binding antigen peptide derived from WT1

ABSTRACT

The present invention provides a method of treating or preventing cancer, by administering a peptide consisting of 16-25 contiguous amino acids in the amino acid sequence of human WT1 of SEQ ID NO: 1, which has the amino acid sequence of SEQ ID NO: 24, an expression vector having a polynucleotide encoding said peptide or a cell containing the expression vector to a subject in need thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a division of U.S. Ser. No. 10/578,183, filedFeb. 27, 2007, which is a National Stage (371) of PCT/JP04/16336, filedNov. 4, 2004, and claims priority to JP 2003-375603, filed Nov. 15,2003.

TECHNICAL FIELD

The present invention relates to HLA-DRB1*0405-binding antigen peptidesderived from WT1.

BACKGROUND ART

WT1 gene (Wilms' tumor gene 1) has been identified as one of causativegenes of Wilms' tumor that is a childhood renal tumor (Cell 60: 509,1990, Nature 343: 774, 1990). WT1 gene encodes the transcription factorWT1 which plays an important role in many processes such asproliferation, differentiation and apoptosis of cells, and developmentof tissues (Int. Rev. Cytol. 181: 151, 1998). WT1 gene was originallydefined as a tumor suppressor gene. However, subsequent studies revealedthat WT1 gene is highly expressed in leukemia and various solid cancersincluding lung cancer and breast cancer, indicating that WT1 gene ratherexerts an oncogenic function that promotes cancer growth. In addition,it was demonstrated that, when peripheral blood mononuclear cellspositive for HLA-A*0201 or HLA-A*2402 were stimulated in vitro withWT1-derived peptides, peptide-specific cytotoxic T-lymphocytes (CTLs)were induced and killed leukemic or solid tumor cells which endogenouslyexpress WT1. These results demonstrated that WT1 is a promising targetmolecule of cancer immunotherapy (Int. J. Hematol 76: 127, 2002).

It has been reported that presence of helper T cells specific to cancerantigen is essential for effective induction of CTLs (Cancer. Res. 62:6438, 2002).

Helper T cells (CD4-positive T cells) are induced (made proliferate) andactivated when they recognize a complex of MHC class II molecule andantigen peptide on antigen-presenting cells. The activated helper Tcells produce cytokines such as IL-2, IL-4, IL-5, IL-6, and/orinterferons and mediate the growth, differentiation, and maturation of Bcells. The activated helper T cells also function to promote the growth,differentiation or maturation of other subsets of T cells such as Tc andTD cells. Thus, the activated helper T cells can activate the immunesystem through the promotion of growth and activation of B and T cells.Therefore, it was suggested to be helpful to enhance functions of helperT cells being under the influence of MHC-class II-binding antigenpeptide (also referred to as “helper peptide”), whereby efficacy(potency) of cancer vaccine in cancer immunotherapy (cancer vaccinetherapy) is increased (J. Immunother., 24:195, 2001).

As for WT1-derived peptides, only one antigen peptide is known to bindto a subtype of MHC class II molecule, i.e., HLA-DRB1*0401 (CancerImmunol. Immunother. 51:271, 2002). There are no WT1-derived peptideswhich have been reported to bind to different subtypes.

DISCLOSURE OF INVENTION

The purpose of the present invention is to provide HLA-DRB1*0405-bindingantigen peptides derived from WT1, and use of the peptide as an enhancerof cancer vaccine efficacy (an agent for enhancing efficacy of cancervaccine).

The present inventor has conducted intensive study on WT1-derivedantigen peptides (“helper peptides”) having an activity of binding toMHC class II antigen and enhancing the cancer vaccine efficacy (potency)in cancer immunotherapy. In consequence, the present inventor has forthe first time found that WT1 contains an antigen peptide portion(s)which has an activity of binding to HLA-DRB1*0405 among a number of MHCclass II subclasses and inducing helper T cells. This finding led to thedevelopment of a novel therapeutic method by which WT1-specific helper Tcells are induced and enhanced in HLA-DRB1*0405-positive cancerpatients.

Recent researches revealed that there exists promiscuous helper peptideswhich are helper peptides capable of binding to plural HLA-class IImolecules and inducing helper CD4-positive T cells (British J. cancer,85(10), p 1527-1534 (2001); J. Immunol., 169, p 557-565 (2002)). Thepresent inventor made investigation into WT1₃₃₂₋₃₄₇, which is one of theabove-described HLA-DRB1*0405-binding antigen peptides (helperpeptides), to elucidate whether or not it is potentially a promiscuoushelper peptide. As a result, said peptide proved to be a promiscuoushelper peptide that binds not only to HLA-DRB1*0405 molecule but also toHLA-DRB1*1502. Thus, the WT1₃₃₂₋₃₄₇ peptide of the present invention isa helper peptide applicable to patients having HLA-DRB1*1502 as well asthose having HLA-DRB1*0405. The present inventor also found that WT1contains an antigen peptide portion(s) capable of binding toHLA-DRB1*1502, one of a number of MHC class II subclasses, and inducinghelper T cells for the first time.

The present invention has been established on the basis of thesefindings.

The present invention encompasses the followings.

(1) A peptide consisting of 10-25 contiguous amino acids in the aminoacid sequence of human WT1 shown in SEQ ID NO: 1, which binds toHLA-DRB1*0405 and induces helper T cells.

(2) The peptide of (1) above, which comprises an amino acid sequence setforth in any one of SEQ ID NOS: 2-23.

(3) The peptide of (2) above, which comprises the amino acid sequenceset forth in SEQ ID NO: 24.

(4) A peptide of 10-25 amino acids, which comprises an amino acidsequence wherein the amino acid residue at position 1, 4, 6 and/or 9 ofan amino acid sequence set forth in any one of SEQ ID NOS: 2-23 issubstituted by another amino acid residue, and which binds to anHLA-DRB1*0405 and induces helper T cells.

(5) The peptide of (4) above, which comprises an amino acid sequencewherein the amino acid residue at position 1, 4, 6 and/or 9 of an aminoacid sequence set forth in any one of SEQ ID NOS: 2-23 is substituted byan amino acid residue selected from the following amino acids:

phenylalanine, tyrosine, tryptophan, valine, isoleucine, leucine andmethionine for the position 1;

valine, isoleucine, leucine, methionine, aspartic acid and glutamic acidfor the position 4;

asparagine, serine, threonine, glutamine, lysine and aspartic acid forthe position 6; and

aspartic acid, glutamic acid and glutamine for the position 9.

(6) The peptide of (5) above, which comprises an amino acid sequencewherein the amino acid residue at position 3, 6, 8 and/or 11 of theamino acid sequence set forth in SEQ ID NO: 24 is substituted by anamino acid residue selected from the following amino acids:

phenylalanine, tryptophan, valine, isoleucine, leucine and methioninefor the position 3;

valine, isoleucine, methionine, aspartic acid and glutamic acid for theposition 6;

asparagine, serine, threonine, glutamine, lysine and aspartic acid forthe position 8; and

aspartic acid, glutamic acid and glutamine for the position 11.

(7) A peptide consisting of 10-25 contiguous amino acids in the aminoacid sequence of human WT1 shown in SEQ ID NO: 1, which binds toHLA-DRB1*1502 and induces helper T cells.

(8) The peptide of (7) above, which comprises an amino acid sequence setforth in any one of SEQ ID NOS: 46-56.

(9) The peptide of (7) above, which comprises the amino acid sequenceset forth in SEQ ID NO: 24.

(10) A peptide comprising a peptide described in any one of (1) to (9)above together with a cancer antigen peptide.

(11) A polynucleotide encoding a peptide described in any one of (1) to(10) above.

(12) An expression vector containing the polynucleotide described in(11) above.

(13) A cell containing the expression vector described in (12) above.

(14) A process for producing a peptide described in any one of (1) to(10) above, which comprises culturing the cell described in (13) aboveunder the condition where the peptide can be expressed.

(15) An antibody which specifically binds to a peptide described in anyone of (1) to (9) above.

(16) A pharmaceutical composition which comprises a peptide described inany one of (1) to (10) above, an expression vector described in (12)above or a cell described in (13) above, in association with apharmaceutically acceptable carrier.

(17) The pharmaceutical composition of (16) above, which is atherapeutic or preventive agent for cancer.

(18) The pharmaceutical composition of (16) above, which is an inducerof helper T cells, and which comprises a peptide described in any one of(1) to (9) above; an expression vector described in (12) related to apeptide of any one of (1) to (9) above; or a cell described in (13)above related to a peptide of any one of (1) to (9) above, inassociation with a pharmaceutically acceptable carrier.

(19) The pharmaceutical composition of (16) above, which is an enhancerof cancer vaccine efficacy, and which comprises a peptide described inany one of (1) to (9) above; an expression vector described in (12)related to a peptide of any one of (1) to (9) above; or a cell describedin (13) above related to a peptide of any one of (1) to (9) above, inassociation with a pharmaceutically acceptable carrier.

(20) The pharmaceutical composition of (16) above, which is atherapeutic or preventive agent for cancer, and which comprises apeptide described in (10) above; an expression vector described in (12)related to a peptide of (10) above; or a cell described in (13) aboverelated to a peptide of (10) above, in association with apharmaceutically acceptable carrier.

(21) Use of a peptide described in any one of (1) to (10) above, anexpression vector described in (12) above or a cell described in (13)above for the manufacture of a therapeutic or preventive agent forcancer.

(22) A method of treating or preventing cancer, which comprisesadministering a peptide described in any one of (1) to (10) above, anexpression vector described in (12) above or a cell described in (13)above to a subject in need thereof.

(23) A pharmaceutical composition which comprises a peptide described inany one of (1) to (9) above in combination with a cancer antigenpeptide.

(24) The pharmaceutical composition of (23) above, which is used fortreating or preventing cancer.

(25) A kit for treating or preventing cancer, which comprises apharmaceutical composition comprising a peptide of any one of (1) to (9)above in association with a pharmaceutically acceptable carrier, and apharmaceutical composition comprising a cancer antigen peptide inassociation with a pharmaceutically acceptable carrier.

(26) Use of a peptide of any one of (1) to (9) above in combination witha cancer antigen peptide in the manufacture of a therapeutic orpreventive agent for cancer.

(27) A method of treating or preventing cancer, which comprisesadministering a peptide of any one of (1) to (9) above in combinationwith a cancer antigen peptide to a subject in need thereof.

The present invention provides an HLA-DRB1*0405-binding antigen peptidederived from WT1, a polynucleotide encoding the peptide, an inducer ofhelper T cells (“helper T cell inducer”) comprising said peptide orpolynucleotide, and the like. The helper T cell inducer of the presentinvention is useful as an enhancer of cancer vaccine efficacy. Theenhancer of cancer vaccine efficacy of the present invention isapplicable to many HLA-DRB1*0405-positive patients, and is particularlyuseful for enhancing efficacy of WT1 vaccine.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the results of examination into responsiveness ofCD4-positive T cells (helper T cells) stimulated with a WT1-derivedWT1₃₃₂₋₃₄₇ peptide to various dendritic cells. In the figure,“Untreated” represents the responsiveness to dendritic cells not pulsedwith a peptide; “PHA” the results of examination wherein CD4 positive Tcells were treated with PHA instead of dendritic cells, “WT1₁₇₂₋₁₈₆pulse” the responsiveness to dendritic cells pulsed with WT1₁₇₂₋₁₈₆peptide, “WT1₂₂₅₋₂₄₃ pulse” the responsiveness to dendritic cells pulsedwith WT1₂₂₅₋₂₄₃ peptide, and “WT1₃₃₂₋₃₄₇ pulse” the responsiveness todendritic cells pulsed with “WT1₃₃₂₋₃₄₇ peptide. The vertical axisindicates the amount of [³H]-thymidine uptake (cpm) by CD4-positive Tcells.

FIG. 2 shows the results of examination into responsiveness of G2 celllines to dendritic cells pulsed with a WT1-derived WT1₃₃₂₋₃₄₇ peptide.In the figure, “Untreated” represents the results obtained usingdendritic cells not pulsed with a peptide; and “WT1₃₃₂₋₃₄₇ pulse” to theresults obtained using dendritic cells pulsed with WT1₃₃₂₋₃₄₇. Thevertical axis indicates the amount of [³H]-thymidine uptake (cpm) by G2cell lines.

FIG. 3 shows the results of examination into responsiveness of G2 cellline to B-LCL(+) cells expressing WT1 gene. In the figure, “B-LCL(−)”represents the results obtained using B-LCL(−) cells not-expressing WT1gene, “B-LCL(+)” the results obtained using B-LCL(+) cells expressingWT1 gene, and “B-LCL(+)+anti-HLA-DR antibody” to the results obtainedusing B-LCL(+) cells treated with anti-HLA-DR antibody. The verticalaxis indicates the amount of [³H]-thymidine uptake (cpm) by G2 celllines.

FIG. 4 shows the results of examination into responsiveness of E04.1cell line to dendritic cells pulsed with a WT1-derived WT1₃₃₂₋₃₄₇peptide. In the figure, “-” represents the results obtained usingdendritic cells not pulsed with a peptide, and “332” the resultsobtained using dendritic cells pulsed with WT1₃₃₂₋₃₄₇ peptide. Thevertical axis indicates the amount of [³H]-thymidine uptake (cpm) byE04.1 cell lines.

FIG. 5 shows the results of examination into responsiveness of E04.1cell line to stimulated cells which have been pulsed with WT1-derivedWT1₃₃₂₋₃₄₇ peptide and then treated with various anti-HLA inhibitoryantibodies. The stimulated cell used is B-LCL(−) cell which is a B cellline established from blood of a healthy volunteer positive forHLA-DRB1*0405 as shown herein below in Example 3. In the figure, “-”represents the results obtained using stimulated cells not pulsed with apeptide, and “332” the results obtained using stimulated cells pulsedwith WT1₃₃₂₋₃₄₇ peptide. Further, “332+α-classI” represents the resultsobtained using stimulated cells treated with WT1₃₃₂₋₃₄₇ peptide andanti-HLA-class I antibody, “332+α-DR” to the results obtained usingstimulated cells treated with the WT1₃₃₂₋₃₄₇ peptide and anti-HLA-DRantibody, “332+α-DQ” to the results obtained using stimulated cellstreated with WT1₃₃₂₋₃₄₇ peptide and anti-HLA-DQ antibody. In addition,“332+mIgG” represents the results obtained using stimulated cellstreated with WT1₃₃₂₋₃₄₇ peptide and anti-mouse-IgG antibody as anegative control for inhibitory antibody. The vertical axis indicatesthe amount of [³H]-thymidine uptake (cpm) by E04.1 cell lines.

FIG. 6 shows the results of examination into responsiveness of E04.1cell line to HLA-DRB1*0405-positive or -negative PBMC pulsed withWT1-derived WT1₃₃₂₋₃₄₇ peptide. In the figure, “-” represents theresults obtained using PBMC not pulsed with a peptide, and “332” theresults obtained using PBMC pulsed with WT1₃₃₂₋₃₄₇ peptide. HLA-DRB1genotypes of respective donors are as follows. Donor 1(HLA-DRB1*0405/0803), Donor 2 (HLA-DRB1*0405/0101), Donor3(HLA-DRB1*0101/1001) and Donor 4 (HLA-DRB1*1201/0802). The vertical axisindicates the amount of [³H]-thymidine uptake (cpm) by E04.1 cell lines.

FIG. 7 shows the results of examination into responsiveness of E04.1cell line to B-LCL(+) cells expressing WT1 gene. In the figure,“B-LCL(−)” represents the results obtained using B-LCL(−) cellnot-expressing WT1 gene as stimulated cells, and “B-LCL(+)” the resultsobtained using B-LCL(+) cells expressing WT1 gene as stimulated cells.The vertical axis indicates the amount of [³H]-thymidine uptake (cpm) byE04.1 cell lines.

FIG. 8 shows the results of examination into responsiveness of E04.1cell line to dendritic cells pulsed with B-LCL(+) cells in whichapoptosis has been induced. In the figure, “apoptotic B-LCL (+)”represents the results obtained using dendritic cells pulsed withB-LCL(+) cells expressing WT1 gene and having been induced apoptosis,and “apoptotic B-LCL (−)” represents the results obtained usingdendritic cells pulsed with B-LCL(−) cells not-expressing WT1 gene andhaving been induced apoptosis. Further, “E04.1+” represents the resultsobtained by cocultivation of E04.1 cells with dendritic cells, and“E04.1-” the results obtained by cocultivation without E04.1 cells. Thevertical axis indicates the amount of [³H]-thymidine uptake (cpm) byE04.1 cell lines.

FIG. 9 shows the results of examination into cytokine production ofE04.1 cell lines to dendritic cells pulsed with WT1-derived WT1₃₃₂₋₃₄₇peptide. In the figure, “-” represents the results obtained usingdendritic cells not pulsed with a peptide, and “332” the resultsobtained using dendritic cells pulsed with WT1₃₃₂₋₃₄₇ peptide. Thevertical axis indicates the percentage (%) of E04.1 cells showing theproduction of IL-4 (blank bar) or IFN-γ (filled bar).

FIG. 10 shows the results of analysis of E04.1 cell line stained withanti-CD4 antibody and anti-CXCR3 antibody by flow cytometer. In thefigure, the horizontal and vertical axes indicate cells positive for CD4and for CXCR3, respectively. The percentage of cells positive for bothCD4 and CXCR3 was 90.1%.

FIG. 11 shows the results of examination into influence of WT1-derivedWT1₃₃₂₋₃₄₇ peptide on the induction of WT1-specific CTLs. PBMCsoriginated in a healthy volunteer (HLA-A*2402/1101, DRB1*0405/0803) werecultured for 7 days under the stimulation conditions of (A) WT1₂₃₅₋₂₄₃peptide (FIG. 11A), (B) WT1₂₃₅₋₂₄₃ peptide+WT1₃₃₂₋₃₄₇ peptide (FIG.11B), (C) WT1₂₃₅₋₂₄₃ peptide+E04.1 cell (FIG. 11C) and (D) WT1₂₃₅₋₂₄₃peptide+WT1₃₃₂₋₃₄₇ peptide+E04.1 cell (FIG. 11D). Then, one half of therecovered cells were analyzed with flow cytometer to obtain thepercentage of WT1₂₃₅₋₂₄₃-specific CTL precursors. The horizontal andvertical axes indicate the percentage of cells positive for CD8 and forWT1₂₃₅₋₂₄₃ peptide/HLA-A*2402, respectively.

FIG. 12 shows the results of examination into influence of WT1-derivedWT1₃₃₂₋₃₄₇ peptide on the activation of WT1-specific CTLs. Another halfof the cells recovered in the experiment mentioned in FIG. 11 werestimulated with WT1₂₃₅₋₂₄₃ peptide for 6 hours, and intracellular IFN-γwas stained. The vertical and horizontal axes indicate the cellspositive for intracellular IFN-γ and for anti-mouse IgG antibody,respectively. The figure shows the results of stimulation with (A)WT1₂₃₅₋₂₄₃ peptide (FIG. 12A), (B) WT1₂₃₅₋₂₄₃ peptide+WT1₃₃₂₋₃₄₇ peptide(FIG. 12B), (C)) WT1₂₃₅₋₂₄₃ peptide+E04.1 cell (FIG. 12C), and (D)WT1₂₃₅₋₂₄₃ peptide+WT1₃₃₂₋₃₄₇ peptide+E04.1 cell (FIG. 12D).

FIG. 13 shows the results of examination into responsiveness ofCD4-positive T cells stimulated with WT1-derived WT1₃₃₂₋₃₄₇ peptide withvarious dendritic cells. In the figure, “-” represents theresponsiveness with dendritic cells not pulsed with a peptide, “332” theresponsiveness with dendritic cells pulsed with WT1₃₃₂₋₃₄₇ peptide,“172” the responsiveness with dendritic cells pulsed with WT1₁₇₂₋₁₈₆peptide, and “225” the responsiveness with dendritic cells pulsed withWT1₂₂₅₋₂₄₃ peptide. The vertical axis indicates the amount of[³H]-thymidine uptake (cpm) by CD4-positive T cells. The symbol “**” and“n.s.” mean that the difference in the test groups is statisticallysignificant or is not, respectively.

FIG. 14 shows the results of T-cell repertoire analysis of CD4-positiveT cells stimulated with WT1-derived WT1₃₃₂₋₃₄₇ peptide. The cells werestained with different antibodies specific for respective Vβ chains ofTCR and analyzed by flow cytometry. In FIG. 14A, the cell population inthe lower right portion of quartered area represents Vβ3-positive cells.In panel FIG. 14B, the cell population in the lower right portion ofquartered area represents Vβ20-positive cells.

FIG. 15 shows the results of examination into the responsiveness ofE15.1 cell line or E15.2 cell line to autologous PBMCs pulsed withWT1-derived WT1₃₃₂₋₃₄₇ peptide. In the figure, “-” represents theresults obtained by using autologous PBMCs not pulsed with a peptide,and “332” the results obtained using autologous PBMCs pulsed withWT1₃₃₂₋₃₄₇ peptide. The vertical axis indicates the amount of[³H]-thymidine uptake (cpm) by separated cell lines. FIG. 15A and FIG.15B show the results obtained using E15.1 cell line and E15.2 cell line,respectively. The symbol “**” means that the difference in the testgroups is statistically significant.

FIG. 16 shows the results of examination into cytokine production ofE15.2 cell line to autologous PBMCs pulsed with WT1-derived WT1₃₃₂₋₃₄₇peptide. In the figure, “-” represents the results obtained usingdendritic cells not pulsed with a peptide, and “332” the resultsobtained using autologous PBMCs pulsed with WT1₃₃₂₋₃₄₇ peptide. Thevertical axis indicates the percentage (%) of E15.2 cells showing theproduction of IL-4 (blank bar) or IFN-γ (filled bar).

FIG. 17 shows the results of examination into the relation between theconcentration of WT1-derived WT1₃₃₂₋₃₄₇ peptide pulsed into autologousPBMCs and the responsiveness of E15.2 cell line. The vertical axisindicates the amount of [³H]-thymidine uptake (cpm) by E15.1 cell lines.The horizontal axis indicates the concentration of WT1₃₃₂₋₃₄₇ peptidepulsed into autologous PBMC.

FIG. 18 shows the results of examination into responsiveness of E15.2cell line to PBMCs positive or negative for HLA-DRB1*1502 pulsed withWT1-derived WT1₃₃₂₋₃₄₇ peptide. In the figure, “-” represents theresults obtained by using PBMCs not pulsed with the WT1₃₃₂₋₃₄₇ peptide,and “332” the results obtained using PBMCs pulsed with the peptide. FIG.18A shows the results obtained using PBMCs from anHLA-DRB1*1502-positive healthy volunteer and FIG. 18B the resultsobtained using PBMCs from an HLA-DRB1*1502-negative healthy volunteer.The vertical axis indicates the amount of [³H]-thymidine uptake (cpm) byE15.2 cell lines. The symbol “**” and “n.s.” mean that the difference inthe test groups is statistically significant or not, respectively.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention provides a peptide consisting of 10-25 contiguousamino acids in the amino acid sequence of human WT1 set forth in SEQ IDNO: 1, said peptide binding to HLA-DRB1*0405 and inducing helper Tcells. The present invention encompasses peptides wherein the N-terminaland/or C-terminal amino acid residue is modified or those wherein aparticular amino acid residue(s) is altered.

Hereinafter, “a peptide that induces helper T cells (or a peptide thatinduces CD4-positive T cells)” may be referred to as “a helper peptide”.

The amino acid sequence of human WT1 set forth in SEQ ID NO: 1 is aknown sequence as described in Cell, 60:509, 1990, and NCBI data base(Accession Nos. XP_034418 and P19544).

The peptide of the present invention is a partial peptide which consistsof 10-25 contiguous amino acids present in the amino acid sequence ofhuman WT1 set forth in SEQ ID NO: 1. The definition of “10-25 aminoacids” is based on the facts that peptides having an activity of bindingto MHC class II generally consist of 10 to 25 amino acids(Immunogenetics, 41: 178-228, 1995, Biochimica et Biophysica Acta 1316,85-101 (1996), Immunology, 96, 1-9 (1999), Peptides, Vol. 19, 179-198(1998), Immunobiology, 5th Edt., 116-117, Garland Publishing (2001)).Preferred peptides are those consisting of 13-17 contiguous amino acidsin the amino acid sequence of human WT1.

The peptide of the present invention can be identified by synthesizing apeptide (candidate peptide) consisting of 10-25 contiguous amino acidsin the amino acid sequence set forth in SEQ ID NO: 1, and assayingwhether or not the peptide is capable of binding to HLA-DRB1*0405 andinducing helper T cells.

The synthesis of a peptide can be conducted according to processesgenerally used in the field of peptide chemistry. Such a method can befound in literatures including Peptide Synthesis, Interscience, NewYork, 1966; The Proteins, Vol. 2, Academic Press Inc., New York, 1976;Peptide Synthesis, Maruzen, Inc., 1975; Peptide-Gosei no Kiso to Jikken,Maruzen, Inc., 1985; and Iyakuhin no Kaihatsu (Zoku), Vol. 14, PeptideSynthesis, Hirokawa-syoten, 1991.

It can be examined whether or not a candidate peptide binds toHLA-DRB1*0405 and induces helper T cells using a method described in,for example, Cancer. Immunol. Immunother. 51: 271 (2002), the methoddescribed in the working Examples, or the method described just below.

Specifically, dendritic cells (adherent cells) are prepared by isolatingperipheral blood mononuclear cells (PBMCs) from a human subject positivefor HLA-DRB1*0405, and removing non-adherent cells. Separately, helper Tcells (CD4-positive T cells) are prepared from the sameHLA-DRB1*0405-positive subject by density gradient centrifugation withFicoll-Paque, and so on.

The above-described dendritic cells are cultured after addition of acandidate peptide, and further cultured with the above-described helperT cells. The helper T cells are then recovered and stimulated severaltimes with dendritic cells pulsed with the candidate peptide in asimilar manner. It is possible to evaluate whether or not helper T cellsare induced (activated) in response to stimulation with a peptide bymeasuring, for example, (1) growth activity of helper T cells or (2)cytokine-producing-activity of helper T cells. Specifically, the growthactivity (1) can be examined by measuring the amount of [³H]-thymidineuptake by helper T cells. The cytokine-producing activity (2) can beexamined by measuring the amount of cytokine such as IFN-γ produced byactivated helper T cells by enzyme enzyme-linked immunosorbent assay(ELISA) or the like.

The amino acid sequence of antigen peptides binding to MHC class I orMHC class II molecule and being presented obeys a certain rule (bindingmotif). There are terminal amino acid residues at the both ends ofpeptides binding to MHC class I molecule which play a significant rolein the binding with MHC class I molecule; however, there are no suchamino acids at either end of peptides binding to MHC class II molecule,and the terminal amino acids do not bind to MHC class II molecule. Sucha peptide rather is accommodated and immobilized (fixed) in apeptide-binding groove longitudinally. The immobilization of a peptidein a peptide-binding groove can be achieved through the binding of theside chains of amino acids constituting the peptide to thepeptide-binding groove and the binding of the main chain of the peptideto the side chains of amino acids well-preserved in the entirepeptide-binding groove for MHC class II molecules. A peptide-bindinggroove has small or large pockets and there is amino acid polymorphismin the amino acid residues constituting the pockets depending on the MHCclass II molecule.

X-ray crystallography obtained so far revealed that side chains of aminoacid residues at positions 1, 4, 6 and 9 of the smallest MHC classII-binding peptide engage with these binding pockets.

The amino acid motif of peptides binding to a pocket(s) ofpeptide-binding groove can be estimated by analyzing the pattern ofamino acid residues commonly found in the binding peptides forrespective MHC class II molecules originated in different alleles. It isconsidered that, because peptides of about 9 amino acids having such amotif is accommodated in the peptide-binding groove in such a mannerthat the both termini protrude from the both sites of groove, there arebasically no limitations regarding the length of peptides which can bindto MHC class II molecules. However, in many cases, a long peptide iscleaved into peptides of 13-17 amino acid length by peptidases(Immunobiology, 5th Edt., 116-117, Garland Publishing (2001)).

Regarding the peptide which has the binding activity for HLA-DRB1*0405,the amino acids at positions 1, 4, 6 and 9 in the HLA(MHC)-bindingdomain consisting of 9-amino acids are expected to have the followingregularities (motifs) (Immunogenetics, 41, 178-228 (1995), Biochimica etBiophysica Acta 1316, 85-101 (1996)).

Position 1: phenylalanine (F), tyrosine (Y), tryptophan (W), valine (V),isoleucine (I), leucine (L) and methionine (M)

Position 4: valine (V), isoleucine (I), leucine (L), methionine (M),aspartic acid (D) and glutamic acid (E)

Position 6: asparagine (N), serine (S), threonine (T), glutamine (Q),lysine (K) and aspartic acid (D)

Position 9: aspartic acid (D), glutamic acid (E) and glutamine (Q)

Recently, it has become possible to search for peptide sequencesexpected to bind to MHC class II antigen using software for predictingMHC class II binding peptides (Propred, Bioinformatics 17: 1236, 2001).

The present invention is based on the finding that WT1 (SEQ ID NO: 1)contains antigen peptide portions which bind to HLA-DRB1*0405 (a kind ofMHC class II) and induce helper T cells for the first time. Examples ofputative HLA-DRB1*0405-binding 9-amino-acid portions in the amino acidsequence of said WT1 include the 9-amino-acid portions derived from WT1as shown in SEQ ID NOS: 2-23. Thus, as a specific embodiment of thepeptide of the present invention, the present invention provides apeptide which comprises an amino acid sequence set forth in any one ofSEQ ID NOS: 2-23, and which binds to HLA-DRB1*0405 and induces helper Tcells.

There are no limitations regarding the length of such a peptide on thecondition that said peptide is a partial peptide of WT1 and comprises anamino acid sequence set forth in any one of SEQ ID NOS: 2-23, and has anactivity of binding to HLA-DRB1*0405 and inducing helper T cells. Asmentioned above, a peptide of about 9 amino acids with a binding motifcan accommodate in a peptide-binding groove with the both ends extrudingfrom the both sites of groove, and hence there are essentially nolimitations regarding the length of the peptides capable of binding toMHC class II molecule. However, long peptides are generally cleaved bypeptidases, and the MHC class II-binding peptides having been reportedso far are about 10-25 amino acids in length (Immunogenetics, 41,178-228 (1995), Biochimica et Biophysica Acta 1316, 85-101 (1996),Immunology, 96, 1-9 (1999), Peptides, Vol. 19, 179-198 (1998),immunobiology, 5th Edt., 116-117, Garland Publishing (2001)). Takingthis into account, the peptides of the present invention preferablyconsist of about 10-25 amino acids, and more preferably about 13-17amino acids.

Accordingly, examples of a preferred embodiment of the peptide of thepresent invention which comprises an amino acid sequence set forth inany one of SEQ ID NOS: 2-23 include partial peptides derived from WT1consisting of 10-25 amino acids (preferably, 13-17 amino-acids), whichcomprise an amino acid sequence set forth in any one of SEQ ID NOS:2-23, and which have an activity of binding to HLA-DRB1*0405 andinducing helper T cells.

Examples of more preferred embodiments include partial peptides derivedfrom WT1 consisting of 10-25 amino acids (preferably, 13-17amino-acids), which comprise an amino acid sequence set forth in SEQ IDNO: 12, and which have an activity of binding to HLA-DRB1*0405 andinducing helper T cells. Examples of still more preferred embodimentsinclude partial peptides derived from WT1 consisting of 16-25 aminoacids (preferably, 16-17 amino-acids), which comprise the amino acidsequence set forth in SEQ ID NO: 24, and which have an activity ofbinding to HLA-DRB1*0405 and inducing helper T cells. The amino acidsequence set forth in SEQ ID NO: 24 represents a 16-amino-acid partialpeptide derived from WT1 and includes the amino acid sequence set fortin SEQ ID NO: 12.

Further preferred embodiment is the peptide consisting of the amino acidsequence set forth in SEQ ID NO: 24.

The peptides of the present invention may be altered as appropriatewithin a range that the activity is maintained. As used herein the“alteration” of amino acid residue means substitution, deletion and/oraddition of amino acid residue(s) (the addition is inclusive of additionof amino acid(s) at the N- and/or C-terminus of a peptide). Thesubstitution of amino acid residue(s) is preferred. When the alterationinvolves substitution of an amino acid residue(s), any number of aminoacid residues at any position can be replaced on the condition that theactivity as a helper peptide is retained. However, since a peptide whichbinds to HLA class II molecule is generally about 10-25 amino acids inlength, the alteration is preferably involves one to several aminoacids.

When altering amino acid residue by substitution, it is preferred thatthe amino acid residue at position 1, 4, 6 and/or 9 of a 9 amino acidpeptide having the structure for the binding motif of HLA-DRB1*0405 issubstituted.

Specific examples of substitution-related peptides of the presentinvention include peptides of 10-25 amino acids, which comprise an aminoacid sequence wherein the amino acid residue at position 1, 4, 6 and/or9 of an amino acid sequence set forth in any one of SEQ ID NOS: 2-23 issubstituted by another amino acid residue, and which bind to anHLA-DRB1*0405 and induce helper T cells.

Preferred examples include peptides of 10-25 amino acids, which comprisean amino acid sequence wherein the amino acid residue at position 1, 4,6 and/or 9 of an amino acid sequence set forth in any one of SEQ ID NOS:2-23 is substituted by an amino acid residue selected from the followingamino acids:

phenylalanine, tyrosine, tryptophan, valine, isoleucine, leucine andmethionine for the position 1;

valine, isoleucine, leucine, methionine, aspartic acid and glutamic acidfor the position 4;

asparagine, serine, threonine, glutamine, lysine and aspartic acid forthe position 6; and

aspartic acid, glutamic acid and glutamine for the position 9, and whichbind to an HLA-DRB1*0405 and induce helper T cells.

For example, the substitution of amino acid residue at position 1, 4, 6and/or 9 can be conducted for the purpose of improving the bindingactivity to HLA-DRB1*0405 or enhancing the activity of theabove-mentioned natural-type helper peptides of the present inventionwhich consist of partial sequences of WT1. The parts other than thesubstituted amino acid at position 1, 4, 6 and/or 9 of a peptide mayremain to have the natural-type sequence (i.e., being kept to have thepartial sequence of WT1), or may be further altered as far as theactivity is retained.

More preferred examples include peptides of 10-25 amino acids, whichcomprise an amino acid sequence wherein the amino acid residue atposition 1, 4, 6 and/or 9 of the amino acid sequence set forth in SEQ IDNO: 12 is substituted by an amino acid residue selected from thefollowing amino acids:

phenylalanine, tryptophan, valine, isoleucine, leucine and methioninefor the position 1;

valine, isoleucine, methionine, aspartic acid and glutamic acid for theposition 4;

asparagine, serine, threonine, glutamine, lysine and aspartic acid forthe position 6; and

aspartic acid, glutamic acid and glutamine for the position 9, and whichbind to an HLA-DRB1*0405 and induce helper T cells.

Still more preferred examples include peptides wherein the amino acidresidue at position 3, 6, 8 and/or 11 of the 16-amino-acid partialpeptide derived from WT1 set forth in SEQ ID NO: 24, which comprises theamino acid sequence set forth in SEQ ID NO: 12, is substituted by anamino acid residue selected from the following amino acids:

phenylalanine, tryptophan, valine, isoleucine, leucine and methioninefor the position 3;

valine, isoleucine, methionine, aspartic acid and glutamic acid for theposition 6;

asparagine, serine, threonine, glutamine, lysine and aspartic acid forthe position 8; and

aspartic acid, glutamic acid and glutamine for the position 11.Preferred examples may include peptides of 16-25 amino acids comprisingthe altered amino acid sequence derived from SEQ ID NO: 24.

The present invention also provides a peptide (so-called an epitopepeptide) comprising a helper peptide (natural- or altered-peptide) ofthe present invention together with a cancer antigen peptide.

Recently, it has been reported that an epitope peptide wherein a cancerantigen peptide(s) (also called as “CTL epitope”) and a helperpeptide(s) (also called as “helper epitope”) are linked together caninduce CTLs efficiently. That is, helper T cells (CD4-positive T cells)activated by a helper peptide exert various activities includinginduction of differentiation and maintenance of CTLs, and activation ofeffectors such as macrophages, etc, and hence are considered to enhancethe CTL-induction by cancer antigens. As a concrete example of a peptidewherein a helper peptide(s) and cancer antigen peptide(s) are linkedtogether, it is reported that a DNA (minigene) encoding an epitopepeptide composed of HBV-originated HLA-A2-restricted antigen peptides (6peptides), HLA-A11-restricted antigen peptides (3 peptides) and a helperpeptide induced in vivo CTLs directed to the respective epitopesefficiently (Journal of Immunology 1999, 162: 3915-3925). Practically, apeptide wherein a CTL epitope (tumor antigen peptide corresponding toposition 280-288 of melanoma antigen gp100) and a helper epitope(tetanus toxin-originated T helper epitope) are linked has beensubjected to clinical test (Clinical Cancer Res., 2001, 7:3012-3024).

Accordingly, as a specific embodiment, the peptides of the presentinvention also include epitope peptides comprising a helper peptide ofthe present invention and a cancer antigen peptide.

As the cancer antigen peptide, any of known cancer antigen peptides canbe used; however, it is preferred to use a cancer antigen peptidederived from WT1 (natural or altered peptide). Concrete examples includeWT1-derived peptides restricted to HLA-A1, -A0201, -A0204, -A0205,-A0206, -A0207, -A11, -A24, -A31, -A6801, -B7, -B8, -B2705, -B37,-Cw0401, -Cw0602, and the like.

Examples of WT1-derived cancer antigen peptides include those listed inTable II-Table XLVI of WO2000/18795 and altered peptides thereof whichhave the activity as a cancer antigen peptide (an activity of binding toan HLA antigen and inducing CTLs).

More concrete examples of WT1-derived cancer antigen peptides includethe followings.

(SEQ ID NO: 27) Cys Met Thr Trp Asn Gln Met Asn Leu (SEQ ID NO: 28)Cys Tyr Thr Trp Asn Gln Met Asn Leu (SEQ ID NO: 29)Arg Met Phe Pro Asn Ala Pro Tyr Leu (SEQ ID NO: 30)Arg Tyr Pro Ser Cys Gln Lys Lys Phe (SEQ ID NO: 31)Ser Tyr Thr Trp Asn Gln Met Asn Leu (SEQ ID NO: 32)Ala Tyr Thr Trp Asn Gln Met Asn Leu (SEQ ID NO: 33)Abu Tyr Thr Trp Asn Gln Met Asn Leu (SEQ ID NO: 34)Arg Tyr Thr Trp Asn Gln Met Asn Leu (SEQ ID NO: 35)Lys Tyr Thr Trp Asn Gln Met Asn Leu (SEQ ID NO: 36)Arg Tyr Phe Pro Asn Ala Pro Tyr Leu (SEQ ID NO: 37)Arg Tyr Pro Gly Val Ala Pro Thr Leu (SEQ ID NO: 38)Ala Tyr Leu Pro Ala Val Pro Ser Leu (SEQ ID NO: 39)Asn Tyr Met Asn Leu Gly Ala Thr Leu (SEQ ID NO: 40)Arg Val Pro Gly Val Ala Pro Thr Leu (SEQ ID NO: 41)Arg Tyr Pro Ser Ser Gln Lys Lys Phe (SEQ ID NO: 42)Arg Tyr Pro Ser Ala Gln Lys Lys Phe (SEQ ID NO: 43)Arg Tyr Pro Ser Abu Gln Lys Lys Phe (SEQ ID NO: 44)Ser Leu Gly Glu Gln Gln Tyr Ser Val (SEQ ID NO: 45)Asp Leu Asn Ala Leu Leu Pro Ala ValIn the above, “Abu” refers to “α-aminobutyric acid.

Among them, the peptides set forth in SEQ ID NOS: 27 and 29 are HLA-A24antigen- and HLA-A2 antigen-binding peptides, and the peptides set forthin SEQ ID NOS: 44 and 45 are HLA-A2 antigen-binding peptides. The otherpeptides are HLA-A24 antigen-binding peptides.

Preferred cancer antigen peptide is the one set forth in SEQ ID NO: 27,28, 29, 30, 44 or 45.

More specific examples of epitope peptides of the present inventioninclude those which comprise a helper peptide that is a WT1-derivedpartial peptide of 10-25 amino acids comprising an amino acid sequenceset forth in any one of SEQ ID NOS: 2-23 and has an activity of bindingto HLA-DRB1*0405 and inducing helper T cells, together with a cancerantigen peptide set forth in any one of SEQ ID NOS: 27-45 above.

Epitope peptides preferably comprise a helper peptide consisting of theamino acid sequence set forth in SEQ ID NO: 24 together with a cancerantigen peptide set forth in any one of SEQ ID NOS: 27-45.

More preferably, epitope peptides comprise a helper peptide consistingof the amino acid sequence set forth in SEQ ID NO: 24 together with acancer antigen peptide set forth in any one of SEQ ID NOS: 27-30, 44 and45.

The epitope peptides can be prepared by aforementioned usual method forpeptide synthesis. It can also be prepared by a usual method for DNAsynthesis and genetic engineering on the basis of sequence informationof a polynucleotide encoding an epitope peptide wherein multipleepitopes are ligated. Specifically, an epitope peptide wherein amultiple epitopes are ligated can be prepared by inserting apolynucleotide encoding the peptide into a known expression vector,transforming a host cell with the resultant recombinant expressionvector, culturing the transformants, and recovering the objectivepeptide from the culture. These processes can be conducted according to,for example, a method described in a literature (Molecular Cloning, T.Maniatis et al., CSH Laboratory (1983), DNA Cloning, D M. Glover, IRLPRESS (1985)), or that described hereinafter.

The activity of said epitope peptide as a helper peptide can beconfirmed according to the above-mentioned method. Further, the activityof said epitope peptide as a cancer antigen peptide can be confirmed bysubjecting the peptide to model animals for human described inWO02/47474 or Int J. Cancer. 100, 565-570, 2002.

An epitope peptide of the present invention is considered to be usefulto establish more efficient treatment or prevention of cancer, because ahelper peptide portion in the helper epitope peptide can activate helperT cells (CD4-positive T cells) to give activated helper T cells whichexert various activities including induction of differentiation andmaintenance of CTLs and activation of effectors such as macrophages,whereby it enhances CTL-induction by cancer antigen peptides further.

The amino group of the N-terminal amino acid or the carboxyl group ofthe C-terminal amino acid of the above-described peptide of the presentinvention (natural-, altered- or epitope-peptide) may be modified. Thepeptides wherein the N-terminal and/or C-terminal amino acid residue ismodified fall within the scope of the peptide of the present invention.

Examples of a group usable in the modification of amino group of theN-terminal amino acid include 1 to 3 groups selected from C₁₋₆ alkylgroup, phenyl group, cycloalkyl group and acyl group. Acyl groupsinclude C₁₋₆ alkanoyl group, C₁₋₆ alkanoyl group substituted by phenylgroup, carbonyl group substituted by C₅₋₇ cycloalkyl group, C₁₋₆alkylsulfonyl group, phenylsulfonyl group, C₂₋₆ alkoxycarbonyl group,alkoxycarbonyl group substituted by phenyl group, carbonyl groupsubstituted by C₅₋₇ cycloalkoxy group, phenoxycarbonyl group, and thelike.

Examples of peptides modified at the carboxyl group of C-terminal aminoacid include esters and amides. Esters specifically include C₁₋₆ alkylesters, C₀₋₆ alkyl esters substituted by phenyl group, C₅₋₇ cycloalkylesters, and the like. Amides specifically include amides, amidessubstituted by one or two C₁₋₅ alkyl groups, amides substituted by oneor two C₀₋₆ alkyl groups that are substituted by phenyl group, amidesforming 5- to 7-membered azacycloalkane inclusive of nitrogen atom ofamide group, and the like.

The present invention also provides a polynucleotide encoding theabove-mentioned peptide (natural-, altered- or epitope-peptide) of thepresent invention. The polynucleotide encoding a peptide of the presentinvention may be in the form of DNA or RNA. The polynucleotides of thepresent invention can be easily prepared on the basis of informationabout amino acid sequence of the present peptide or polynucleotidesequence of DNA encoding the same. Specifically, synthesis can becarried out using usual method of DNA synthesis or amplification by PCR.

Concrete examples of polynucleotides include those encoding theabove-mentioned epitope peptides. More specifically, examples ofpolynucleotide include those encoding epitope peptides which comprise ahelper peptide that is a WT1-derived partial peptide of 10-25 aminoacids comprising an amino acid sequence set forth in any one of SEQ IDNOS: 2-23 and has an activity of binding to HLA-DRB1*0405 and inducinghelper T cells, together with a cancer antigen peptide set forth in anyone of SEQ ID NOS: 27-45 above.

Preferred examples include a polynucleotide encoding an epitope peptidewhich comprises a helper peptide consisting of the amino acid sequenceset forth in SEQ ID NO: 24 and a cancer antigen peptide set forth in anyone of SEQ ID NOS: 27-45.

More preferred examples include a polynucleotide encoding an epitopepeptide which comprise a helper peptide consisting of the amino acidsequence set forth in SEQ ID NO: 24 and a cancer antigen peptide setforth in any one of SEQ ID NOS: 27-30, 44 and 45.

The “polynucleotide encoding the peptide of the present invention”encompasses polynucleotides that can hybridize under the stringentconditions to the complementary sequence of the said polynucleotide andthat encode peptides which have activities equivalent to the peptide ofthe present invention. In relation to “hybridize under the stringentcondition”, the “hybridization” herein used can be carried out accordingto conventional method described in, for example, Sambrook J., Frisch E.F., Maniatis T. ed. Molecular Cloning 2nd edition, Cold Spring HarborLaboratory press. The term “stringent conditions” means thathybridization is conducted in a solution containing 6×SSC (10×SSC=1.5 MNaCl, 1.5 M trisodium citrate), 50% formamide at 45° C., followed bywashing in a solution of 2×SSC at 50° C. (Molecular Biology, John Wiley& Sons, N.Y. (1989), 6.3.1-6.3.6)), or the like.

A recombinant expression vector for expressing the peptide of thepresent invention can be constructed by incorporating a polynucleotideprepared above into an expression vector.

As expression vectors usable herein include plasmids, phage vectors,virus vectors, and the like.

When the host is Escherichia coli, examples of vector include plasmidvectors such as pUC118, pUC119, pBR322, pCR3, and the like; and phagevectors such as λZAPII, Agt11, and the like. When the host is yeast,examples of vector include pYES2, pYEUra3, and the like. When the hostis insect cells, examples of vector include pAcSGHisNT-A, and the like.When the host is animal cells, examples of vector include plasmidvectors such as pKCR, pCDM8, pGL2, pcDNA3.1, pRc/RSV, pRc/CMV, and thelike; and virus vectors such as retrovirus vector, adenovirus vector,adeno-associated virus vector, and the like.

The expression vector may optionally contain a factor(s) such aspromoter capable of inducing expression, a gene encoding a signalsequence, a marker gene for selection, terminator, and the like.

Furthermore, the expression vector may contain an additional sequencefor allowing the peptide to express as a fusion protein withthioredoxin, His tag, GST (glutathione S-transferase), or the like, soas to facilitate the isolation and purification. Vectors usable in sucha case include GST fusion protein vectors containing an appropriatepromoter (lac, tac, trc, trp, CMV, SV40 early promoter, etc) thatfunctions in host cells, such as pGEX4T; vectors containing Tag sequence(Myc, His, etc) such as pcDNA3.1/Myc-His; and vectors capable ofexpressing a fusion protein between thioredoxin and His tag such aspET32a.

Transformed cells containing the vector of the present invention can beprepared by transforming host cells with an expression vector obtainedin the above.

Host cells usable herein include Escherichia coli, yeast, insect cellsand animal cells. Examples of Escherichia coli include strains of E.coli K-12 such as HB101, C600, JM109, DH5α and AD494 (DE3). Examples ofyeast include Saccharomyces cerevisiae. Examples of animal cells includeL929, BALB/c3T3, C127, CHO, COS, Vero and Hela cells. Examples of insectcells include sf9.

Introduction of an expression vector into host cells can be done using aconventional method suited for the respective host cells above.Specifically, introduction can be done using calcium phosphate method,DEAE-dextran method, electroporation method, and a method using lipidfor gene transfer (Lipofectamine, Lipofectin; Gibco-BRL). Following theintroduction, the cells are cultured in a conventional medium containinga selection marker, whereby transformants containing the expressionvector can be selected.

The peptide of the present invention can be produced by culturing thetransformed cells under appropriate conditions (conditions under whichpeptides can be expressed). The resultant peptide may be furtherisolated and purified according to standard biochemical purificationprocedures. The purification procedures include salting out, ionexchange chromatography, absorption chromatography, affinitychromatography, gel filtration chromatography, etc. When the polypeptideof the present invention has been expressed as a fusion peptide withthioredoxin, His tag, GST, or the like, as mentioned above, the peptidecan be isolated and purified by appropriate purification proceduresmaking use of the characteristics of the fusion protein or tags.

The present invention provides an antibody which specifically binds to apeptide of the present invention. The antibody of the present inventionis not restricted to any form and may be polyclonal or monoclonalantibody raised against a peptide of the present invention as anantigen.

As mentioned above, there is no limitation regarding the antibody of thepresent invention on the condition that it specifically binds to apeptide of present invention. Examples of preferred antibody includethose specifically bind to a helper peptide that is a WT1-derivedpartial peptide of 10-25 amino acids comprising an amino acid sequenceset forth in any one of SEQ ID NOS: 2-23 and has an activity of bindingto HLA-DRB1*0405 and inducing helper T cells. Antibodies specificallybind to a helper peptide consisting of the amino acid sequence set forthin SEQ ID NO: 24 is more preferred.

Methods of preparation of antibodies are well known in the art and theantibodies of the present invention can be prepared according to any oneof conventional methods (Current protocols in Molecular Biology edit.Ausubel et al. (1987) Publish. John Wiley and Sons. Section 11.12-11.13,Antibodies; A Laboratory Manual, Lane, H, D. et al., ed., Cold SpringHarber Laboratory Press, New York 1989).

Specifically, antibodies of the present invention can be obtained byimmunizing a non-human animal such as rabbit using a peptide of thepresent invention as an antigen, and recovering the antibodies fromserum of the immunized animal in a conventional manner. In the case ofmonoclonal antibodies, they can be obtained by immunizing a non-humananimal such as mouse with a peptide of the present invention, subjectingthe resultant splenocytes to cell fusion with myeloma cells, andrecovering monoclonal antibodies from the resultant hybridoma cells(Current protocols in Molecular Biology edit. Ausubel et al. (1987)Publish. John Wiley and Sons. Section 11.4-11.11).

The antibodies against the peptide of the present invention can also beproduced while enhancing the immunological response using differentadjuvants depending on the host. Examples of adjuvants include Freundadjuvants; mineral gels such as aluminium hydroxide; surfactants such aslysolecithin, Pluronic® polyol, polyanion, peptide, oil emulsion,keyhole limpet hemocyanin and dinitorophenol; human adjuvants such asBCG (Bacille de Calmette-Guerin) or Corynebacterium, etc.

As mentioned above, antibodies that recognize a peptide of the presentinvention and antibodies that neutralize the activity thereof can easilybe prepared by immunizing an animal in a conventional manner. Theantibodies may be used in affinity chromatography, immunologicaldiagnostic method, and the like. Immunological diagnostic method may beselected as appropriate from immunoblotting, radioimmunoassay (RIA),enzyme-linked immunosorbent assay (ELISA), a fluorescent or luminescentassay, and the like. The immunological diagnostic method is effective inthe diagnosis of cancer expressing WT1 gene such as gastric cancer,colon cancer, lung cancer, breast cancer, embryonal cancer, skin cancer,bladder cancer, prostate cancer, uterine cancer, cervical cancer,ovarian cancer, and the like.

The present invention provides a pharmaceutical composition comprising apeptide (natural-, altered-, and epitope-type peptides) of the presentinvention, an expression vector containing a polynucleotide of thepresent invention or a cell containing an expression vector of thepresent invention, in association with a pharmaceutically acceptablecarrier. The pharmaceutical composition can be used effectively as aninducer of helper T cells or an enhancer of cancer vaccine efficacy, asdescribed in detail below.

(1) An Inducer of Helper T Cells Comprising as an Active Ingredient aPeptide of the Present Invention (an Enhancer of Cancer VaccineEfficacy)

The peptide of the present invention has an activity of inducing helperT cells, and the induced helper T cells in turn are able to enhance theCTL-inducing activity, which is the effects of cancer vaccine, throughthe induction of differentiation and maintenance of CTLs and theactivation of effectors such as macrophages. Thus, the present inventionprovides an enhancer of cancer vaccine efficacy comprising as an activeingredient a peptide of the present invention (pharmaceuticalcomposition as an agent for enhancing efficacy of cancer vaccine). Whenthe enhancer of the present invention is administered to anHLA-DRB1*0405-positive and WT1-positive patient, the peptide ispresented to HLA-DRB1*0405 antigen of an antigen-presenting cell;specific helper T cells (CD4-positive T cells) recognizing a complex ofthe peptide and HLA-DRB1*0405 antigen are induced and activated; and theactivated helper T cells can exert the activity concerning induction ofdifferentiation and maintenance of CTLs and activation of effectors suchas macrophages. In this manner, the activity of activating and inducingCTLs as the effect of cancer vaccine is enhanced.

The enhancer of cancer vaccine efficacy of the present invention can beused in the prevention or treatment of cancer accompanied by elevatedexpression level of WT1 gene, for example, blood cancers such asleukemia, myelodysplastic syndrome, multiple myeloma and malignantlymphoma, and solid cancers such as gastric cancer, colon cancer, lungcancer, breast cancer, embryonal cancer, hepatic cancer, skin cancer,bladder cancer, prostate cancer, uterine cancer, cervical cancer, andovarian cancer.

The enhancer of cancer vaccine efficacy of the present invention can beadministered concurrently with, before or after the administration ofcancer vaccine.

The enhancer of cancer vaccine efficacy comprising as an activeingredient a peptide of the present invention may contain as an activeingredient a helper peptide(s) or an epitope peptide wherein apeptide(s) is ligated with a cancer antigen peptide(s) (CTL epitope(s))As mentioned above, it has recently been shown that an epitope peptidewherein a cancer antigen peptide (CTL epitope) and a helper peptide(helper epitope) can induce CTLs efficiently. When an epitope peptide ofthis form is administered, said peptide is incorporated intoantigen-presenting cells; among the antigen peptides generated byintracellular degradation, helper peptides bind to MHC class II antigen(HLA-DRB1*0405) while cancer antigen peptides to MHC class I antigen;and respective complexes thus formed are presented on the surface ofantigen-presenting cells in high density. When helper T cells recognizethe complex of HLA-DRB1*0405 antigen and helper peptide, theCTL-inducing activity that is the effect of cancer vaccine is furtherenhanced as a result of induction of differentiation and maintenance ofCTLs and the activation of effectors such as macrophages. On the otherhand, when CTLs recognize the complex of cancer antigen peptides and MHCclass I antigen, CTLs proliferate and destroy the cancer cells. Thus,the pharmaceutical composition of the present invention comprising as anactive ingredient an epitope peptide of the present invention can beused as a cancer vaccine per se, as well as an enhancer of cancervaccine efficacy.

The enhancer of cancer vaccine efficacy which comprises as an activeingredient a peptide of the present invention may be administeredtogether with a pharmaceutically acceptable carrier, for example, anappropriate adjuvant, or in the form of particles so that the cellularimmunity can be established effectively. As an adjuvant, those describedin a literature (Clin. Microbiol. Rev., 7:277-289, 1994), and the likeare applicable. Concrete examples include microorganism-derivedcomponents, cytokines, plant-derived components, marine organism-derivedcomponents, mineral gels such as aluminium hydroxide, surfactants suchas lysolecithin and Pluronic® polyols, polyanions, peptides, oilemulsion (emulsion preparations) and the like. Liposomal preparations,particulate preparations in which the ingredient is bound to beadshaving a diameter of several μm, preparations in which the ingredient isattached to lipids, and the like, are also contemplated.

Administration may be achieved, for example, intradermally,subcutaneously, intramuscularly, or intravenously. Although the dosageof the peptide of the present invention in the formulation may beadjusted as appropriate depending on, for example, the disease to betreated, the age and the body weight of a patient, it is usually withinthe range of 0.0001 mg-1000 mg, preferably 0.001 mg-1000 mg, morepreferably 0.1 mg-10 mg, which can be preferably administered once inevery several days to every several months.

The present invention also provides a pharmaceutical compositioncomprising a combination of a peptide of the present invention and acancer antigen peptide. According to the combined use of the presentinvention, the effect of a cancer antigen peptide as cancer vaccine(i.e., activity of inducing and activating CTLs) is enhanced by thepeptide of the present invention, and the treatment or prevention ofcancer can be achieved more effectively.

The term “combination” encompasses the both forms where a peptide of thepresent invention and a cancer antigen peptide are administered in amixed form or discrete forms.

Administration of the peptides in the mixed form can be conducted usinga previously prepared formulation containing the peptides as a mixture,or combining previously prepared formulations each comprising therespective peptides before use.

In the case where peptides are administered in discrete forms, thediscrete formulations may be administered successively with atime-interval, or administered concurrently. When administration isconducted with a time-interval, a peptide of the present invention(enhancer of cancer vaccine efficacy) and a cancer antigen peptide(cancer vaccine) may be administered in this order or in reverse order.

An embodiment of the combination of a peptide of the present inventionand a cancer antigen peptide includes a kit.

As a cancer antigen peptide usable in combination with a peptide of thepresent invention, any cancer antigen peptides conventionally known canbe used, and examples include WT1-derived cancer antigen peptides(natural-, altered-type). Concrete examples include a cancer antigenpeptide set forth in any one of SEQ ID NOS: 27-45, preferably, SEQ IDNOS: 27-30, 44 and 45.

(2) Inducer of Helper T Cells (Enhancer of Cancer Vaccine Efficacy)Comprising as an Active Ingredient an Expression Vector of the PresentInvention

An expression vector containing a polynucleotide encoding a peptide ofthe present invention, similar to the above-mentioned peptide of thepresent invention, has an activity of inducing helper T cells and isuseful as an active ingredient of an enhancer of cancer vaccine efficacyof the present invention. Thus, the present invention provides anenhancer of cancer vaccine efficacy (i.e., a pharmaceutical compositionas an agent enhancing efficacy of cancer vaccine) comprising as anactive ingredient an expression vector containing a polynucleotideencoding a peptide of the present invention.

Recently, a polynucleotide encoding an epitope peptide wherein a cancerantigen peptide (CTL epitope) and a helper peptide (helper epitope) areligated has been shown to have an activity of inducing CTLs in vivoefficiently. For example, it is reported that a DNA (minigene) encodingan epitope peptide wherein HBV-originated HLA-A2-restricted antigenpeptides (6 peptides), HLA-A11-restricted antigen peptides (3 peptides)and a helper epitope are ligated induced in vivo CTLs directed to therespective epitopes efficiently (Journal of Immunology 1999, 162:3915-3925).

Accordingly, an active ingredient of enhancer of cancer vaccine efficacycan be obtained by incorporating a polynucleotide encoding theabove-described epitope peptide of present invention into an appropriateexpression vector.

When administering an expression vector containing a polynucleotide ofthe present invention as an active ingredient of enhancer of cancervaccine efficacy, the following methods can be used.

As a method for introducing an expression vector containing thepolynucleotide of the present invention into cells, any means includingthose utilizing viral vectors or other methods are applicable(Nikkei-Science, April, 1994, p 20-45; Gekkan-Yakuji, 36(1), p 23-48(1994); Jikken-Igaku-Zokan, 12(15), 1994, and references cited therein).

Examples of means utilizing a viral vector include those wherein a DNAof the present invention is incorporated into DNA or RNA virus such asretrovirus, adenovirus, adeno-associated virus, herpes virus, vacciniavirus, poxvirus, poliovirus, or Sindbis virus, and then introduced intocells. Above all, a method utilizing retrovirus, adenovirus,adeno-associated virus, or vaccinia virus, or the like, is particularlypreferred.

Examples of other methods include those wherein an expression vector isdirectly injected intramuscularly (DNA vaccination), liposome method,Lipofectin method, microinjection, calcium phosphate method andelectroporation. DNA vaccination and liposome method are particularlypreferred.

Regarding a method to make the expression vector of the presentinvention act as a medicament in practice, there are an in vivo methodwherein the expression vector is directly introduced into the body andan ex vivo method wherein the expression vector is introducedextracorporeally into a certain cells removed from human, and the cellsare reintroduced into the body (Nikkei-Science, April, 1994, 20-45;Gekkan-Yakuji, 36(1), 23-48 (1994); Jikkenn-Igaku-Zokan, 12(15), 1994;and references cited therein). The in vivo method is more preferred.

In the case of in vivo method, the administration can be effectedthrough any appropriate routes depending on the disease and symptoms tobe treated. For example, it may be administered via intravenous,intraarterial, subcutaneous, intracutaneous, intramuscular route, or thelike. When the administration is carried out by in vivo method, thecompositions may be administered in various forms such as solution, andare typically formulated, for example, in the form of injectioncontaining, as an active ingredient, an expression vector of the presentinvention to which conventional carriers may also be added, ifnecessary. As for the liposomes or membrane-fused liposomes (such asSendai virus (HVJ)-liposomes) containing an expression vector of thepresent invention, they may be in the form of liposomal formulation suchas suspension, frozen drug, centrifugally-concentrated frozen drug, orthe like.

Although the content of an expression vector in a formulation may beadjusted as appropriate depending on, for example, the disease to betreated, age and body weight of a patient, usually, 0.0001 mg-100 mg,preferably 0.001 mg-10 mg of an expression vector of the presentinvention can be administered once in every several days to everyseveral months.

When the above-mentioned expression vector of the present invention isadministered to an HLA-DRB1*0405-positive and WT1-positive patient, thepeptide of the present invention is presented to HLA-DRB1*0405 antigenof an antigen-presenting cell; specific helper T cells (CD4-positive Tcells) recognizing a complex of the peptide and HLA-DRB1*0405 antigenare induced and activated; the activated helper T cells can exert theactivity concerning induction of differentiation and maintenance of CTLsand activation of effectors such as macrophages. In this manner, theactivity of inducing CTLs as an effect of cancer vaccine is enhanced.The enhancer of cancer vaccine efficacy comprising as an activeingredient an expression vector containing a polynucleotide of thepresent invention can be used in the prevention or treatment of canceraccompanied by elevated expression level of WT1 gene, for example, bloodcancers such as leukemia, myelodysplastic syndrome, multiple myeloma andmalignant lymphoma, and solid cancers such as gastric cancer, coloncancer, lung cancer, breast cancer, embryonal cancer, hepatic cancer,skin cancer, bladder cancer, prostate cancer, uterine cancer, cervicalcancer, and ovarian cancer.

In the above case where an expression vector containing a polynucleotideencoding an epitope peptide is administered, antigen presenting cellsincorporate the same and generate antigen peptides through intracellulardegradation, of which helper peptides and cancer antigen peptides bindto MHC class II antigen (HLA-DRB1*0405) and MHC class I antigen,respectively, to form complexes. The so formed complexes are presentedon the surface of antigen presenting cells in high density. When helperT cells recognize the complex of HLA-DRB1*0405 antigen and helperpeptide, the CTL-inducing activity that is the effect of cancer vaccineis further enhanced through the induction of differentiation andmaintenance of CTLs and the activation of effectors such as macrophages.On the other hand, when CTLs recognize the complex of cancer antigenpeptides and MHC class I antigen, CTLs proliferate and destroy thecancer cells. Thus, the pharmaceutical composition of the presentinvention comprising as an active ingredient an expression vectorcontaining a polynucleotide encoding an epitope peptide of the presentinvention can be used as a cancer vaccine per se, as well as an enhancerof cancer vaccine efficacy.

The present invention also provides a peptide consisting of 10-25contiguous amino acids in the amino acid sequence of human WT1 set forthin SEQ ID NO: 1, said peptide binding to HLA-DRB1*1502 and inducinghelper T cells. The present invention encompasses antigen peptidesbinding to HLA-DRB1*1502 wherein the N-terminal and/or C-terminal aminoacid residue is modified or those wherein a particular amino acidresidue(s) is altered.

The synthesis and determination of activity of antigen peptides bindingto HLA-DRB1*1502 of the present invention can be carried out in a mannersimilar to that described in relation to the above-mentioned antigenpeptides binding to HLA-DRB1*0405 of the present invention.

The HLA-DRB1*1502-binding antigen peptides of the present invention is apartial peptide which consists of 10-25 contiguous amino acids in theamino acid sequence of human WT1 set forth in SEQ ID NO: 1. Preferredpeptides are those consisting of 13-17 contiguous amino acids in theamino acid sequence of human WT1.

The present invention is based on the finding that human WT1 contains anantigen peptide portion having activity of binding to HLA-DRB1*1502 andinducing helper T cells. Search for 9-amino-acid portions potentiallybinding to HLA-DRB1*1502 (9-amino-acid portions capable of beingaccommodated in a peptide-binding groove of MHC class II molecule) wasconducted using a software for predicting MHC class II binding peptides(Propred, Bioinformatics 17: 1236, 2001). Examples of the identified9-amino-acid portions of WT1 are shown in SEQ ID NOS: 46-56. Thus,specific examples of HLA-DRB1*1502-binding antigen peptides of thepresent invention include a peptide which comprises an amino acidsequence set forth in any one of SEQ ID NOS: 46-56, and which binds toHLA-DRB1*1502 and induces helper T cells.

The said peptides preferably consist of about 10-25 amino acids, andmore preferably about 13-17 amino acids. Examples of more preferredembodiments include partial peptides derived from WT1 consisting of10-25 amino acids (preferably, 13-17 amino-acids), which comprise theamino acid sequence set forth in SEQ ID NO: 50, and which have anactivity of binding to HLA-DRB1*1502 and inducing helper T cells.Examples of still more preferred embodiments include partial peptidesderived from WT1 consisting of 16-25 amino acids (preferably, 16-17amino-acids), which comprise the amino acid sequence set forth in SEQ IDNO: 24, and which have an activity of binding to HLA-DRB1*1502 andinducing helper T cells. The amino acid sequence set forth in SEQ ID NO:24 represents a 16-amino-acid partial peptide derived from WT1 andincludes the amino acid sequence set fort in SEQ ID NO: 50.

Further preferred embodiment is the WT1₃₃₂₋₃₄₇ peptide consisting of theamino acid sequence set forth in SEQ ID NO: 24.

The said WT1₃₃₂₋₃₄₇ peptide is a promiscuous helper peptide which bindsnot only to HLA-DRB1*0405 molecule but also to HLA-DRB1*1502 molecule.Accordingly, WT1₃₃₂₋₃₄₇ is a helper peptide applicable to patientshaving HLA-DRB1*0405 and those having HLA-DRB1*1502 as well, and henceis useful from the viewpoint of wide application range of patients.

The epitope peptide, polynucleotide, antibody, and pharmaceuticalcomposition regarding the HLA-DRB1*1502-binding antigen peptide of thepresent invention can be made and use (put into effect) in a mannersimilar to above-mentioned HLA-DRB1*0405-binding antigen peptide of thepresent invention.

EXAMPLES

The present invention is further illustrated by the following examples,but should not be construed as being limited thereto.

Example 1

1. Preparation of Dendritic Cells

Peripheral blood mononuclear cells (PBMCs) were isolated by densitygradient centrifugation with Ficoll-Paque from blood of anHLA-DRB1*0405-positive healthy volunteer. The resultant 8×10⁶ PBMCs weresuspended in 2 ml of X-VIVO 15™ medium (Camblex) containing 1% AB serum,seeded in a 6-well culture plate, and cultured for 2 hours. Aftercultivation, non-adherent cells were removed, and the adherent cellswere washed with Hanks solution. Adherent cells were cultured in X-VIVO15™ medium containing 1% AB serum, 1000 U/ml IL-4 and 1000 U/ml GM-CSF.On days 2 and 4 of cultivation, one-half of medium was replaced by freshmedium. On day 6, TNF-α was added to make the final concentration of 100U/ml. Cells existing on day 7 were used as dendritic cells in theexperiment.

2. Preparation of CD4-Positive T Cells (Helper T Cells)

Blood obtained from the same healthy volunteer as in (1) above was used.Blood was diluted by 2-times with RPMI medium. To about 100 ml of thediluted blood was added antibody cocktail, RosetteSep™ (Stemcell) forseparation of CD4-positive T cells, and the mixture was left to stand atroom temperature for 20 minutes. CD4-positive T cells were thencollected by density gradient centrifugation with Ficoll-Paque.

3. Induction of WT1 Peptide-Specific CD4-Positive T Cells

Amino acid sequence of WT1 protein (NCBI database, Accession No. P19544,XP_034418, SEQ ID NO: 1) was searched for peptides potentially bindingto HLA-DRB1*0405 using a prediction program (Propred, Bioinformatics 17:1236, 2001). Three peptides were selected and synthesized. Thesepeptides have the same amino acid sequences as those present at thefollowing positions of WT1:

Position 172-186: (WT1₁₇₂₋₁₈₆, SEQ ID NO: 25) PNHSFKHEDPMGQQG;Position 225-243: (WT1₂₂₅₋₂₄₃, SEQ ID NO: 26) NLYQMTSQLECMTWNQMNL; andPosition 332-347: (WT1₃₃₂₋₃₄₇, SEQ ID NO: 24) KRYFKLSHLQMHSRKH.

Dendritic cells prepared in (1) above were seeded in 24-well cultureplate at 3×10⁵ cells/well, and a peptide of SEQ ID NO: 24 was added upto 50 μg/ml. After 4-hour-cultivation, cell growth was stopped by X-rayirradiation (25 Gy). CD4-positive cells prepared in (2) above were addedto each well at 3×10⁶ cells/well and cocultured with dendritic cells. Asmedium, X-VIVO 15™ medium containing 1% AB serum was used. After thecultivation was started, one half of medium was replaced with freshmedium every 2 days and IL-2 was added up to 20 U/ml. On days 7 and 14from the beginning of cultivation, T cells were collected and seeded in24-well plate at 3×10⁶ cells/well, and thereto were added 3×10⁵dendritic cells having been pulsed with 20 μg/ml of a peptide (SEQ IDNO: 24) and undergone X-ray irradiation (25 Gy). The cells were thencocultured. As medium, X-VIVO 15™ medium containing 1% AB serum and 20U/ml IL-2 was used.

After the third stimulation, T cells were recovered and seeded in96-well plate at 3×10⁴ cells/well. Dendritic cells having been pulsedwith 20 μg/ml of a peptide (SEQ ID NO: 24) and undergone X-rayirradiation (25 Gy) were added at 3×10⁴ cells/well, followed bycoculture. As a negative control group, T cells were cocultured withdendritic cells not pulsed with the peptide, and as a positive controlgroup, 0.2% PHA was added instead of dendritic cells. After80-hour-cultivation, [³H]-thymidine (37 kBq/well) was added, and thecells were cultured for another 16 hours. [³H]-thymidine incorporated bycells was then measured using β-scintillation counter. The results areshown in FIG. 1. CD4-positive T cells stimulated with a peptide atposition 332-347 of WT1 (WT1₃₃₂₋₃₄₇, SEQ ID NO: 24) showed proliferativeresponse when cocultured with dendritic cells pulsed with WT1₃₃₂₋₃₄₇.However, CD4-positive T cells did not show proliferative response whencocultured with dendritic cells not pulsed with the peptide, ordendritic cells pulsed with a peptide having amino acid sequence of SEQID NO: 25 or 26 which is different from that of SEQ ID NO: 24. Theseresults demonstrate that the WT1₃₃₂₋₃₄₇ peptide (SEQ ID NO: 24) inducesspecific CD4-positive T cells as an antigen peptide.

Example 2

Establishment of CD4-Positive T-Cell Lines Specific for WT1 Peptides

Dendritic cells prepared in a manner similar to Example 1 were seeded in96-well plate at 10⁴ cells/well, and then CD4-positive T cells inducedby WT1₃₃₂₋₃₄₇ peptide (SEQ ID NO: 24) were seeded at 10³ cells/well. Asmedium, X-VIVO 15™ medium containing 1% AB serum, 20 U/ml IL-2 and 5μg/ml PHA was used. CD4-positive T-cell line was established bycontinued cultivation and named as “G2 cell line”. Responsiveness of G2cell line to dendritic cells pulsed with a peptide was measured by asimilar method to Example 1. The results are shown in FIG. 2. G2 cellline showed proliferative response when cocultured with dendritic cellspulsed with WT1₃₃₂₋₃₄₇ peptide, but did not when cocultured withdendritic cells not-pulsed with the peptide.

These results demonstrate that G2 cell line is a CD4-positive T-cellline specific for WT1₃₃₂₋₃₄₇ peptide.

Example 3

Antigen Presentation of WT1 Peptide to HLA-DR Molecule

Peripheral blood mononuclear cells (PBMCs) were isolated by densitygradient centrifugation with Ficoll-Paque from blood of anHLA-DRB1*0405-positive healthy volunteer in a manner similar toExample 1. PBMCs were then seeded in 24-well plate at 10⁷ cells/well. Asmedium, RPMI1640 medium containing 10% FCS and 55 μM 2ME was used. Afteradding medium containing Epstein-Barr virus (EBV), cultivation wascontinued for another 4 weeks to establish B-cell line transformed withEBV, which cell line was named as “B-LCL(−) cell”. EBV was prepared fromculture supernatant of B95-8 (JCRB Cell Bank No. 9123), a cell line thatproduces EBV. B-LCL(−) cells were adjusted to 3×10⁷ cells/mL, andthereto were added medium containing virus expressing WT1 gene and thenpolypropylene (final concentration, 8 μg/mL), and the mixture was addedto a 24-well plate at 1 ml/well. After 16-hour-cultivation, 1 ml offresh medium was added to each well, and cultivation was continued. Toeach well was added G418 (neomycin) up to 0.7 μg/mL, and the plate wascultured for 5 to 7 days, when cells into which the gene was introducedwere selected. The selected B-cell line expressing WT1 was named as“B-LCL(+) cell”. The amount of WT1 gene expressed by B-LCL(−) andB-LCL(+) cells was measured by RT-PCR technique according to the methoddescribed in Blood, 89:1405, 1997. The measurements were converted byassuming the expression amount of K562 cell line as the positive controlto be 1. The resulting value for B-LCL(−) cells was 1.6×10⁻⁴ while thatfor B-LCL(+) cells 3.2, indicating that WT1 gene is highly expressed.Responsiveness of G2 cells to B-LCL(+) cells was examined in a mannersimilar to Example 2. In a test group, B-LCL(+) cells were treated withanti-HLA-DR antibody before mixing with G2 cells to confirm theHLA-DR-restriction. The results are shown in FIG. 3. It was revealedthat G2, which is a CD4-positive T-cell line positive for the peptide,shows proliferative response when cocultured with B-LCL(+) cellsexpressing endogenous WT1 genes, and that said response is inhibited byanti-HLA-DR antibody. These results demonstrate that WT1₃₃₂₋₃₄₇ peptideis intracellularly generated from WT1 protein and endogenously presentedas an antigen to HLA-DR molecule.

Example 4

Establishment of CD4-Positive T-Cell Lines E04.1 Specific for WT1₃₃₂₋₃₄₇Peptide

Dendritic cells were prepared using blood isolated from anHLA-DRB1*0405-positive healthy volunteer in a manner similar to Example1 except that the final concentration of TNF-α added on day 6 was 200IU/ml. CD4-positive T cells were prepared using blood obtained from thesame healthy volunteer as that used for the preparation of dendriticcells. CD4-positive T cells were separated according to the instructionsof RosetteSep (StemCell) for separation of CD4-positive T cells.

The above-described dendritic cells and CD4-positive T cells were usedto induce CD4-positive T cells specific for a WT1 peptide (SEQ ID NO:24, WT1₃₃₂₋₃₄₇) in a manner similar to Example 1. The resultantCD4-positive T cells specific for WT1₃₃₂₋₃₄₇ peptide were culturedcontinuously by limiting dilution technique to establish CD4-positiveT-cell line E04.1. As feeder cells in the limiting dilution technique,PBMCs prepared in a manner similar to Example 1 and treated by X-rayirradiation were seeded at 1×10⁵ cells/well. As medium, X-VIVO 15™medium containing 20 IU/ml IL-2 and 5 μg/ml PHA was used.

Responsiveness of E04.1 cell line to dendritic cells pulsed withWT1₃₃₂₋₃₄₇ peptide was measured by a similar method to Example 1 exceptthat cultivation was continued for 18 hours after adding [³H]-thymidine.The results are shown in FIG. 4. E04.1 cells showed proliferativeresponse when cocultured with dendritic cells pulsed with WT1₃₃₂₋₃₄₇peptide but did not when cocultured with dendritic cells not-pulsed withthe peptide. These results demonstrate that E04.1 cell is a CD4-positiveT-cell line specific for WT1₃₃₂₋₃₄₇ peptide.

Example 5

Specific Binding of WT1₃₃₂₋₃₄₇ Peptide to HLA-DR

E04.1 cells established in Example 4 were seeded in 96-well cultureplate at 1×10⁴ cells/well. B cell line B-LCL (−) cells established fromblood of an HLA-DRB1*0405-positive healthy volunteer in Example 3 werepulsed with WT1₃₃₂₋₃₄₇ peptide at a concentration of 20 μg/ml andtreated by X-ray irradiation, seeded in 96-well plate at 3×10⁴cells/well, and cocultured with E04.1 cells. As a negative controlgroup, B-LCL (−) cells not pulsed with the peptide were cocultured withE04.1 cells.

As test groups, B-LCL (−) cells having been pulsed with WT1₃₃₂₋₃₄₇peptide and undergone X-ray irradiation were treated with 20 μg/ml ofanti-HLA-DR antibody (G46.6, BD ParMingen), anti-H LA-class I antibody(G46-2.6, BD ParMingen), or anti-HLA-DQ antibody (SPVL3, Immunotech) for30 minutes, and cocultured with E04.1 cells to confirm HLA-DR restrictednature. As a negative control group for antibody treatment, cellstreated with anti-mouse IgG antibody were cocultured with E04.1 cells ina similar manner.

Following cocultivation, growth of E04.1 cells was measured in a mannersimilar to Example 4. The results are shown in FIG. 5. E04.1 cellsshowed proliferative response when cocultured with B-LCL (−) cellspulsed with WT1₃₃₂₋₃₄₇ peptide. However, when WT1₃₃₂₋₃₄₇ peptide-pulsedB-LCL (−) cells were treated with anti-HLA-DR antibody, growth of cellswas inhibited. Further, E04.1 cells showed proliferative response toWT1₃₃₂₋₃₄₇-peptide-pulsed B-LCL (−) cells treated with other antibody,but did not show proliferative response to B-LCL (−) cells not pulsedwith the peptide. These results demonstrate that WT1₃₃₂₋₃₄₇ peptidespecifically binds to HLA-DR among HLA molecules, and induces growth ofCD4-positive E04.1 cell line specific for WT1₃₃₂₋₃₄₇ peptide.

Example 6

Specific Binding of WT1₃₃₂₋₃₄₇ Peptide to HLA-DRB1*0405

PBMCs were prepared from blood of HLA-DRB1*0405-positive or -negativehealthy volunteer in a manner similar to Example 4. PBMCs were thenpulsed with 20 μg/ml of WT1₃₃₂₋₃₄₇ peptide and subjected to X-rayirradiation, and seeded in 96-well plate at 3×10⁴ cells/well. E04.1cells were then seeded into the 96-well plate at 1×10⁴ cells/well, andthe cells were cocultured. As a negative control group, PBMCs not pulsedwith the peptide and E04.1 cells were cocultured.

Following cocultivation, growth of E04.1 cells was measured in a mannersimilar to Example 4. The results are shown in FIG. 6. Donor 1(HLA-DRB1*0405/0803) and Donor 2 (HLA-DRB1*0405/0101) areHLA-DRB1*0405-positive, and E04.1 cells cocultured with PBMCs isolatedfrom each donor and pulsed with WT1₃₃₂₋₃₄₇ peptide showed proliferativeresponse. On the other hand, Donor 3 (HLA-DRB1*0101/1001) and Donor 4(HLA-DRB1*1201/0802) are HLA-DRB1*0405-negative, and E04.1 cellscocultured with PBMCs isolated from each donor and pulsed withWT1₃₃₂₋₃₄₇ peptide did not show proliferative response. Further, in allcases, no proliferative response was observed when PBMCs not-pulsed withthe peptide were used. The above results demonstrate that WT1₃₃₂₋₃₄₇peptide specifically binds to HLA-DRB1*0405 among HLA-DRB1 moleculesshowing polymorphism, and induce the growth of WT1₃₃₂₋₃₄₇-specificCD4-positive cell line E04.1.

Example 7

Antigen Presentation of WT1₃₃₂₋₃₄₇ Peptide to HLA-DRB1*0405

B-LCL(−) cells (B-cell line) and B-LCL (+) cells (B-cell line expressingWT1), which were established from blood of an HLA-DRB1*0405-positivehealthy volunteer as described in Example 3, were each subjected toX-ray irradiation and seeded in 96-well plate at 3×10⁴ cells/well. E04.1cells were seeded into each well at 1×10⁴ cells and cocultured. Growthresponse of E04.1 cells was then measured in a manner similar to Example4. The results are shown in FIG. 7. E04.1 cells showed proliferativeresponse when cocultured with B-LCL (+) cells expressing WT1, but notwhen cocultured with B-LCL (−) cells not-expressing WT1.

Next, B-LCL(−) or B-LCL (+) cells (1×10⁵ cells each) having been inducedapoptosis were cocultured for 16 hours with dendritic cells (3×10⁴cells) prepared from blood of an HLA-DRB1*0405-positive healthyvolunteer in a manner similar to Example 4, seeded in well of 96-wellplate, and cocultured with E04.1 cells (1×10⁴ cells). Growth response ofE04.1 cells was then measured in a manner similar to Example 1. Theresults are shown in FIG. 8. E04.1 cells showed proliferative responsewhen cocultured with dendritic cells pulsed with apoptosis-induced B-LCL(+) cells, but did not when cocultured with dendritic cells pulsed withapoptosis-induced B-LCL (−) cells. These results indicate thatWT1₃₃₂₋₃₄₇ peptide is first generated through the degradation of WT1protein in B-LCL (+) cells, then presented to HLA-DRB1*0405 and induceproliferation of E04.1 cells.

Induction of apoptosis in B-LCL (−) and B-LCL (+) cells was conducted byosmotic shock. Namely, 1×10⁶ cells were suspended in 500 μl of ahyperosmotic medium (RPMI medium containing 0.5 M sucrose, 10% w/vpolyethylene glycol 1000 and 10 mM HEPES, pH 7.2), and left to stand at37° C. for 10 minutes. The culture was then diluted by 30-times with ahypoosmotic medium (60% RPMI, 40% water) previously adjusted to 37° C.,and left to stand at 37° C. for 2-3 minutes. Cells were collected bycentrifuging at room temperature for 5 minutes and used asapoptosis-induced cells. Induction of apoptosis was confirmed by afluorescent dye for staining dead cells (Propidium Iodide, and AnnexinV,i.e., a phosphatidyl serine-binding reagent).

Example 8

Activation of E04.1 Cells with WT1₃₃₂₋₃₄₇ Peptide

Dendritic cells prepared from blood of an HLA-DRB1*0405-positive healthyvolunteer in a manner similar to Example 4 were pulsed with WT1₃₃₂₋₃₄₇peptide, mixed with E04.1 cells, and cultured for 24 hours. As anegative control group, dendritic cells not pulsed with the peptide weremixed with E04.1 cells. After 24-hour-cultivation, Brefeldin A was addedto a final concentration of 10 μg/ml to inhibit exocytosis of E04.1cells. Further, CD4-positive T cells were recovered after culturing foranother 6 hours, and fixed with PBC containing 2% formaldehyde, andtreated by permeabilization solution containing 0.1% saponin to increasethe cell membrane permeability of antibody. The treated cells were thenintracellularly stained by treating with PE-labeled anti-IFN-γ antibody(BD PharMingen) and FITC-labeled anti-IL-4 antibody (BD PharMingen), andanalyzed using a flow cytometer. The results are shown in FIG. 9. It wasrevealed that E04.1 cells, when cocultured with dendritic cells pulsedwith WT1₃₃₂₋₃₄₇ peptide, were induced strongly to produce IFN-γ which isa Th-1-type cytokine, but not to produce IL-4 which is a Th-2-typecytokine.

Unstimulated E04.1 cells were stained by treating with anti-CD4 antibodyand anti-CXCR3 antibody, and analyzed by flow cytometer. The results areshown in FIG. 10. It was revealed that more than 90% of E04.1 cells areCD4-positive T cells of Th-1 type which are positive for CD4 and CXCR3.CXCR3 is known to be a chemokine receptor highly expressed on Th-1 typeimmunocytes.

The above results indicate that WT1₃₃₂₋₃₄₇ peptide activate E04.1 cells,WT1₃₃₂₋₃₄₇-specific CD4-positive cell line, and induce the cells toproduce IFN-γ which is a Th-1 type cytokine. These results demonstratethat WT1₃₃₂₋₃₄₇ peptide activates and makes CD4-positive T cellsdifferentiate into Th-1 type.

Example 9

Enhancement of Induction and Activation of WT1-Specific CTLs byWT1₃₃₂₋₃₄₇ Peptide

PBMCs were prepared using blood of the same healthy volunteer(HLA-A*2402/1101, DRB1*0405/0803) as that used for establishment ofE04.1 cells in a manner similar to Example 4, and seeded in 24-wellculture plate at 3×10⁴ cells/well. To the well were added WT1₂₃₅₋₂₄₃peptide (SEQ ID NO: 27) and E04.1 cells in the following manners, andthe plate was cultured at 37° C. for 7 days. WT1₂₃₅₋₂₄₃ peptide (20μg/ml); WT1₂₃₅₋₂₄₃ peptide (20 μg/ml)+WT1₃₃₂₋₃₄₇ peptide (20 μg/ml);WT1₂₃₅₋₂₄₃ peptide (20 μg/ml)+E04.1 cells (1.5×10⁶ cells/well); orWT1₂₃₅₋₂₄₃ peptide (20 μg/ml)+WT1₃₃₂₋₃₄₇ peptide (20 μg/ml)+E04.1 cells(1.5×10⁶ cells/well). As medium, X-VIVO 15™ medium containing 10% ABserum was used. WT1₂₃₅₋₂₄₃ peptide used here is a cancer antigen peptidehaving activity of inducing HLA-A′2402-restricted CTLs (WO2004/024175).

After 7-day-cultivation, cells were recovered and one half of the cellswere stained using anti-CD8 antibody (BD PharMingen) and WT1₂₃₅₋₂₄₃peptide/HLA-A*2402-specific PE-labeled tetramer, and analyzed by flowcytometer. The results are shown in FIG. 11 (A-D). It has been reportedthat stimulation of PBMCs with WT1₂₃₅₋₂₄₃ peptide leads to induction ofWT1₂₃₅₋₂₄₃ peptide-specific CTL precursors positive for both CD8 andWT1₂₃₅₋₂₄₃ peptide/HLA-A*2402 (Cancer Immunol Immunother, 51, p 614-620(2002)). When PBMCs were stimulated with WT1₂₃₅₋₂₄₃ peptide alone, thepercentage of WT1₂₃₅₋₂₄₃ peptide-specific CTL precursors was 0.12% (FIG.11-A). When stimulation was conducted using WT1₂₃₅₋₂₄₃ peptide plusWT1₃₃₂₋₃₄₇ peptide, the percentage of WT1₂₃₅₋₂₄₃ peptide-specific CTLprecursors increased to 0.69% (FIG. 11-B). When stimulation wasconducted using WT1₂₃₅₋₂₄₃ peptide plus E04.1 cells, the percentage ofWT1₂₃₅₋₂₄₃ peptide-specific CTL precursors further increased to 4.51%(FIG. 11-C). When stimulation was conducted using WT1₂₃₅₋₂₄₃ peptideplus WT1₃₃₂₋₃₄₇ peptide plus E04.1 cells, the percentage of WT1₂₃₅₋₂₄₃peptide-specific CTL precursors still further increased to 7.12% (FIG.11-D).

Another half of the recovered cells (3×10⁵ cells) were cocultured for 6hours with dendritic cells pulsed with WT1₂₃₅₋₂₄₃ peptide and undergoneX-ray irradiation (30 Gy). One hour after the initiation of cultivation,Brefeldin A was added to inhibit exocytosis of cells. Cultivation wascontinued for another 5 hours and the cells were stained with anti-CD8antibody and WT1₂₃₅₋₂₄₃ peptide/HLA-A*2402-specific PE-labeled tetramer.The cells were fixed and treated with a permeabilization solution toincrease cell membrane permeability, and intracellularly stained byPE-labeled anti-IFN-γ antibody in a manner similar to Example 8. As anegative control group, cells were stained with APC-labeled anti-mouseIgG antibody (BD PharMingen), and cell populations positive for bothIFN-γ and mouse IgG were excluded as non-specific staining.

The results are shown in FIG. 12 (A-D). When WT1₂₃₅₋₂₄₃ peptide-specificCTL precursors are stimulated with WT1₂₃₅₋₂₄₃ peptide for 6 hours, CTLswhich are specific for activated WT1₂₃₅₋₂₄₃ peptide and are positive forCD8, WT1₂₃₅₋₂₄₃ peptide/HLA-A*2402 and IFN-γ are induced. Whenstimulation was conducted using WT1₂₃₅₋₂₄₃ peptide alone, the percentageof CTLs specific for activated WT1₂₃₅₋₂₄₃ peptide was 17.0% (FIG. 12-A).When stimulation was conducted using WT1₂₃₅₋₂₄₃ peptide plus WT1₃₃₂₋₃₄₇peptide, the percentage of CTLs specific for activated WT1₂₃₅₋₂₄₃peptide was increased to 23.3% (FIG. 12-B). When stimulation wasconducted using WT1₂₃₅₋₂₄₃ peptide plus E04.1 cells, the percentage ofCTLs specific for activated WT1₂₃₅₋₂₄₃ peptide was increased to 25.7%(FIG. 12-C). When stimulation was conducted using WT1₂₃₅₋₂₄₃ peptideplus WT1₃₃₂₋₃₄₇ peptide plus E04.1 cells, the percentage of CTLsspecific for activated WT1₂₃₅₋₂₄₃ peptide was increased to 39.0% (FIG.12-D).

From the above results, it was revealed that WT1₃₃₂₋₃₄₇ peptide is ahelper peptide which enhances the induction and activation ofWT1-specific CTL precursors. It was also revealed that E04.1 cell is ahelper T cell which enhances the activation of WT1-specific CTLs andthat its helper function is increased by WT1₃₃₂₋₃₄₇ peptide and theactivation of WT1-specific CTLs is enhanced.

Example 10

Investigation into Promiscuous Nature of WT1₃₃₂₋₃₄₇ Peptide

WT1₃₃₂₋₃₄₇ peptide was examined whether it also can bind toHLA-DRB1*1502 molecule, which is said that many Japanese possess, andinduce WT1₃₃₂₋₃₄₇-specific CD4-positive T cells as a promiscuous helperpeptide.

1. Experimental Method

1) Preparation of Dendritic Cells (DC)

Peripheral blood mononuclear cells (PBMCs) were isolated from peripheralblood of a healthy volunteer (HLA-DRB1*1502/1403), and seeded in 6-wellplastic plate using 1% AB-type serum (Nabi, Miami, Fla.) and X-VIVO15™medium (Cambrex) at 1×10⁷ cells/well, and cultured for 2 hours. Afterremoving non-adherent cells, the remaining adherent cells were culturedin a medium containing 1000 IU/ml IL-4(PeproTech), 1000 IU/ml GM-CSF(PeproTech), 1% AB-type serum, and X-VIVO 15™ medium. On days 2 and 4,medium was changed and IL-4 and GM-CSF were added, and on day 6, TNF-αwas added up to 100 IU/ml to make dendritic cells mature.

2) Induction of WT1₃₃₂₋₃₄₇-Specific CD4-Positive T Cells

CD4-positive T cells were isolated from blood of the same volunteerusing RosetteSep (StemCell) for separating CD4-positive T cells. Theresulting CD4-positive T cells were seeded in 24-well plate at 3×10⁶cells/well, and stimulated with autologous dendritic cells (3×10⁵ cells)pulsed with WT1₃₃₂₋₃₄₇ peptide (20 μg/ml) and undergone radiationirradiation (25 Gy). On the next day from stimulation, IL-2 was added upto 20 IU/ml. In a similar manner, the stimulated CD4-positive T cellswere stimulated every one week with dendritic cells pulsed withWT1₃₃₂₋₃₄₇ peptide (20 μg/ml). Further, medium change was conductedusing IL-2-containing medium on every other day following the secondstimulation. In the experiments, CD4-positive T cells induced by 3 timesof stimulation in total.

3) Growth Assay

Growth assay was conducted by [³H]-thymidine incorporation method.CD4-positive T cells induced by stimulation with a peptide (3×10⁴ cells;responder) were cocultured with PBMCs pulsed with a peptide selectedfrom WT1₃₃₂₋₃₄₇, WT1₁₇₂₋₁₈₆ and WT1₂₂₅₋₂₄₃ peptides and undergoneradiation irradiation (1×10⁵ cells, “stimulator”) in a 96-well plate. Asa negative control, DC(−) not pulsed with a peptide was used. After80-hour-cocultivation, [³H]-thymidine (Amersham Biosciences) was addedat 37 kBq/well. The plate was incubated for another 16 hours andmeasured with β scintillation counter. Measurement unit is “count perminute (cpm)”, and every assay was carried out in triplicate.

4) Analysis of Cytokine Production by Flow Cytometry

In the same manner as growth assay, CD4-positive T cells were coculturedwith stimulators for 2 hours, and thereto was added Brefeldin A. Fourhours later, cells were recovered, subjected to treatment for fixationand permeation, stained with FITC-labeled anti-IL-4 antibody (BDPharmingen) and PE-labeled anti-IFN-γ antibody (BD Pharmingen), andanalyzed by flow cytometry.

5) ELISA Assay

PBMCs (6×10⁵ cells) pulsed or not pulsed with WT1₃₃₂₋₃₄₇ peptide weretreated by radiation irradiation (25 Gy), and each group of cells wascocultured with WT1₃₃₂₋₃₄₇-induced CD4-positive T cells (6×10⁵ cells).After 72-hour-cultivation, the supernatant was recovered, and 300 μl ofthe solution was used for measurement of IL-4 and IFN-γ.

6) TCR Repertoire Assay

Repertoire of β chain of T-cell receptor (TCR) for CD4-positive T cellsinduced by WT1₃₃₂₋₃₄₇ peptide was analyzed using TCR Vβ repertoire Kit(BECKMAN COULTER), FACsort (BECTON DICKINSON).

2. Experimental Results

CD4-positive T cells isolated from blood of an healthy volunteer(HLA-DRB1*1502/1403) were stimulated three times in total withautologous dendritic cells pulsed with WT1₃₃₂₋₃₄₇ peptide. The soinduced CD4-positive T cells were examined for the peptide specificityby growth assay using each of WT1₁₇₂₋₁₈₆, WT1₂₂₅₋₂₄₃ and WT1₃₃₂₋₃₄₇peptides. As a result, CD4-positive T cells induced by WT1₃₃₂₋₃₄₇peptide did not proliferate in the absence of the peptide or under thestimulation with WT1₁₇₂₋₁₈₆ or WT1₂₂₅₋₂₄₃, but proliferated to becomeabout 10-times as much when stimulated with WT1₃₃₂₋₃₄₇ (FIG. 13). Fromthese results, the induced CD4-positive T cells have specificity forWT1₃₃₂₋₃₄₇.

The WT1₃₃₂₋₃₄₇ peptide-induced CD4-positive T cells were subjected toTCR repertoire assay. The results are shown in FIG. 14. Cells having Vβ3and those having Vβ20 were dominant and each accounted for 10% of thetotal.

These dominant CD4-positive T cells were separated by sorting and thecell line having Vβ3 was named as E15.1 subline, while the one havingVβ20 as E15.2 subline. Of these two cell lines, E15.2 subline showedhigher responsiveness to WT1₃₃₂₋₃₄₇ peptide (FIG. 15).

E15.2 subline was then stimulated with WT1₃₃₂₋₃₄₇ peptide, and thesecreted IL-4 and IFN-γ were measured by Intracellular stain method. Asa result, it was revealed that IFN-γ (Th-1-type cytokine) and not IL-4(Th-2-type cytokine) is dominantly produced by said cell line (FIG. 16).It was also revealed that the WT1₃₃₂₋₃₄₇-specific proliferative-responseof E15.2 subline depends on the concentration of WT1₃₃₂₋₃₄₇ (FIG. 17).These results indicated that E15.2 subline is a Th-1-type CD4-positive Tcell line specific for WT1₃₃₂₋₃₄₇.

As mentioned above, it was possible to induce Th-1-type CD4-positive Tcell lines specific for WT1₃₃₂₋₃₄₇. from CD4-positive T cells derivedfrom a healthy volunteer who is positive for HLA-DRB1*1502 molecule butnegative for HLA-DRB1*0405 molecule. Based on these results, it isconsidered that said CD4-positive T cells participate in cellularimmunity and can activate CTLs through the secretion of cytokines. Thus,it was shown that antitumor effects can be further enhanced by usingWT1₃₃₂₋₃₄₇ in combination with HLA-class I-restricted WT1 peptide(cancer antigen peptide) capable of activating CTLs.

PBMCs derived from an HLA-DRB1*1502-positive healthy volunteer(1502/0901) or an HLA-DRB1*1502-negative healthy volunteer (1302/0803)were pulsed with WT1₃₃₂₋₃₄₇ peptide to obtain stimulators. Each ofstimulators was cocultured with E15.2 subline, and analyzed forWT1₃₃₂₋₃₄₇-specific proliferation by growth assay. The results are shownin FIG. 18. In an HLA-DRB1*1502-positive healthy volunteer,WT1₃₃₂₋₃₄₇-specific proliferation was observed but in anHLA-DRB1*1502-negative healthy volunteer, no proliferation was observed(FIG. 18). This indicated that the WT1₃₃₂₋₃₄₇-specific proliferation ofE15.2 subline is restricted to HLA-DRB1*1502.

As described above, analysis conducted using E15.2 subline which is aTh-1-type CD4-positive T cell line specific for WT1₃₃₂₋₃₄₇ peptidedemonstrated that WT1₃₃₂₋₃₄₇ is a promiscuous helper peptide which bindsnot only to HLA-DRB1*0405 molecule but also to HLA-DRB1*1502 moleculewhich molecules are found in the first and the third frequencies,respectively, among Japanese.

INDUSTRIAL APPLICABILITY OF THE INVENTION

The present invention provides a WT1-derived HLA-DRB1*0405-bindingantigen peptide, a polynucleotide encoding said peptide, a helper T cellinducer comprising said peptide or polynucleotide, and the like. Thehelper T cell inducer of the present invention is useful as an enhancerof cancer vaccine efficacy. The enhancer of cancer vaccine efficacy ofthe present invention is applicable to many cancer patients positive forHLA-DRB1*0405, and particularly useful as an enhancer of WT1 vaccineefficacy.

The invention claimed is:
 1. A method of treating WT1- andHLA-DRB1*0405-expressing cancer or a WT1- and HLA-DRB1*1502-expressingcancer in an HLA-DRB1*0405- or an HLA-DRB1*1502-positive subject, whichcomprises administering a peptide consisting of the amino acid sequenceof SEQ ID NO: 24, an expression vector comprising a polynucleotideencoding a peptide consisting of the amino acid sequence of SEQ ID NO:24 or a cell comprising an expression vector comprising a polynucleotideencoding a peptide consisting of the amino acid sequence of SEQ ID NO:24 to a subject in need thereof.
 2. The method of claim 1, wherein saidWT1-expressing cancer is a blood cancer selected from the groupconsisting of leukemia, myelodysplastic syndrome, multiple myeloma, andmalignant lymphoma.
 3. The method of claim 1, wherein saidWT1-expressing cancer is a solid cancer selected from the groupconsisting of gastric cancer, colon cancer, lung cancer, breast cancer,embryonal cancer, hepatic cancer, skin cancer, bladder cancer, prostatecancer, uterine cancer, cervical cancer, and ovarian cancer.
 4. Themethod of claim 1, wherein said administering is intradermally,subcutaneously, intramuscularly, or intravenously.
 5. A method oftreating WT1- and HLA-DRB1*0405-expressing cancer or a WT1- andHLA-DRB1*1502-expressing cancer in an HLA-DRB1*0405- or anHLA-DRB1*1502-positive subject, which comprises administering a peptideconsisting of 16-25 contiguous amino acids in the amino acid sequence ofhuman WT1 of SEQ ID NO: 1 to a subject in need thereof, wherein saidpeptide comprises the amino acid sequence of SEQ ID NO:
 24. 6. Themethod of claim 5, wherein said WT1-expressing cancer is a blood cancerselected from the group consisting of leukemia, myelodysplasticsyndrome, multiple myeloma, and malignant lymphoma.
 7. The method ofclaim 5, wherein said WT1-expressing cancer is a solid cancer selectedfrom the group consisting of gastric cancer, colon cancer, lung cancer,breast cancer, embryonal cancer, hepatic cancer, skin cancer, bladdercancer, prostate cancer, uterine cancer, cervical cancer, and ovariancancer.
 8. The method of claim 5, wherein said administering isintradermally, subcutaneously, intramuscularly, or intravenously.
 9. Themethod of claim 5, wherein said peptide consists of the amino acidsequence of SEQ ID NO:
 24. 10. The method of claim 1, wherein saidmethod is a method of treating WT1- and HLA-DRB1*0405-expressing cancerin an HLA-DRB1*0405-positive subject.
 11. The method of claim 1, whereinsaid method is a method of treating WT1- and HLA-DRB1*1502-expressingcancer in an HLA-DRB1*1502-positive subject.
 12. The method of claim 5,wherein said method is a method of treating WT1- andHLA-DRB1*0405-expressing cancer in an HLA-DRB1*0405-positive subject.13. The method of claim 5, wherein said method is a method of treatingWT1- and HLA-DRB1*1502-expressing cancer in an HLA-DRB1*1502-positivesubject.
 14. A method of treating WT1-expressing cancer in anHLA-DRB1*0405- or an HLA-DRB1*1502-positive subject, which comprisesadministering a peptide consisting of the amino acid sequence of SEQ IDNO: 24, an expression vector comprising a polynucleotide encoding apeptide consisting of the amino acid sequence of SEQ ID NO: 24 or a cellcomprising an expression vector comprising a polynucleotide encoding apeptide consisting of the amino acid sequence of SEQ ID NO: 24, saidpeptide, expression vector or cell being administered in combinationwith an HLA-class I restricted WT1 peptide capable of activating CTLs toa subject in need thereof.
 15. The method of claim 14, wherein saidWT1-expressing cancer is a blood cancer selected from the groupconsisting of leukemia, myelodysplastic syndrome, multiple myeloma, andmalignant lymphoma.
 16. The method of claim 14, wherein saidWT1-expressing cancer is a solid cancer selected from the groupconsisting of gastric cancer, colon cancer, lung cancer, breast cancer,embryonal cancer, hepatic cancer, skin cancer, bladder cancer, prostatecancer, uterine cancer, cervical cancer, and ovarian cancer.
 17. Themethod of claim 14, wherein said administering is intradermally,subcutaneously, intramuscularly, or intravenously.
 18. The method ofclaim 14, wherein said method is a method of treating WT1- andHLA-DRB1*0405-expressing cancer in an HLA-DRB1*0405-positive subject.19. The method of claim 14, wherein said method is a method of treatingWT1- and HLA-DRB1*1502-expressing cancer in an HLA-DRB1*1502-positivesubject.
 20. A method of treating WT1-expressing cancer in anHLA-DRB1*0405- or an HLA-DRB1*1502-positive subject, which comprisesadministering a peptide consisting of 16-25 contiguous amino acids inthe amino acid sequence of human WT1 of SEQ ID NO: 1 to a subject inneed thereof, wherein said peptide comprises the amino acid sequence ofSEQ ID NO: 24, said peptide being administered in combination with anHLA-class I restricted WT1 peptide capable of activating CTLs to asubject in need thereof.
 21. The method of claim 20, wherein saidWT1-expressing cancer is a blood cancer selected from the groupconsisting of leukemia, myelodysplastic syndrome, multiple myeloma, andmalignant lymphoma.
 22. The method of claim 20, wherein saidWT1-expressing cancer is a solid cancer selected from the groupconsisting of gastric cancer, colon cancer, lung cancer, breast cancer,embryonal cancer, hepatic cancer, skin cancer, bladder cancer, prostatecancer, uterine cancer, cervical cancer, and ovarian cancer.
 23. Themethod of claim 20, wherein said administering is intradermally,subcutaneously, intramuscularly, or intravenously.
 24. The method ofclaim 20, wherein said peptide consists of the amino acid sequence ofSEQ ID NO:
 24. 25. The method of claim 20, wherein said method is amethod of treating WT1- and HLA-DRB1*0405-expressing cancer in anHLA-DRB1*0405-positive subject.
 26. The method of claim 20, wherein saidmethod is a method of treating WT1- and HLA-DRB1*1502-expressing cancerin an HLA-DRB1*1502-positive subject.